The Relative Recoverability Of Dna And Rna Profiles From Forensically Relevant Body Fluid Stains

Transcription

1 University of Central Florida Electronic Theses and Dissertations Masters Thesis (Open Access) The Relative Recoverability Of Dna And Rna Profiles From Forensically Relevant Body Fluid Stains 2011 Charly Parker University of Central Florida Find similar works at: University of Central Florida Libraries Part of the Chemistry Commons, and the Forensic Science and Technology Commons STARS Citation Parker, Charly, "The Relative Recoverability Of Dna And Rna Profiles From Forensically Relevant Body Fluid Stains" (2011). Electronic Theses and Dissertations This Masters Thesis (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of STARS. For more information, please contact

2 THE RELATIVE RECOVERABILITY OF AND RNA PROFILES FROM FORENSICALLY RELEVANT BODY FLUID STAINS by CHARLY PARKER B.S. Berry College, 2008 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Forensic Science in the Department of Chemistry in the College of Sciences at the University of Central Florida Orlando, Florida Spring Term 2011

3 2011 Charly Parker ii

4 ABSTRACT Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mrna profiling to be utilized in routine forensic casework, RNA of sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current analysis methodologies. Several /RNA co-extraction methods were evaluated based on the quantity and quality of and RNA recovered and were also compared to standard non-co-extraction methods. The two most promising methods, the in-house developed NCFS co-extraction and the commercially available AllPrep /RNA Mini kit, were then optimized by improving nucleic acid recovery and consistency of CE (capillary electrophoresis) detection results. The sensitivity of the two methods was also evaluated, and and RNA profiles could be obtained for the lowest amount of blood (0.2 µl) and saliva and semen (1 µl) tested. Both extraction methods were found to be acceptable for use with forensic samples, and the ability to obtain full profiles was not hindered by the co-extraction of RNA. It is generally believed that RNA is less stable than which may prevent its use in forensic casework. However, the degradation rates of and RNA in the same biological fluid stain have not been directly compared. To determine the relative stability of and RNA, the optimized NCFS co-extraction protocol was used to isolate and RNA from iii

5 environmentally compromised stains. Dried blood, saliva, and semen stains and vaginal secretions swabs were incubated at set temperatures and outside for up to 1 year. Even at 56 C, and RNA were both stable out to 1 year in the blood and semen stains, out to 3 months () and 1 year (RNA) in the saliva stains, and out to 6 months () and 3 months (RNA) in the vaginal secretions swabs. The recoverability of both nucleic acids was reduced when the samples were exposed to increased humidity, sunlight, and rain. In general, and RNA stability was found to be similar with a loss in ability to obtain a or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no profile was obtained, indicating that RNA in dried stains is sufficiently stable for mrna body fluid typing to be used in forensic casework. iv

6 To my mother and late father. Thank you for your constant love and support. v

7 ACKNOWLEDGMENTS I would first and foremost like to thank Dr. Jack Ballantyne for giving me the opportunity to be a part of his laboratory and learn more than I could have hoped. I feel lucky to have worked with someone who has as much expertise and knowledge as you do. I would also like to thank Dr. Erin Hanson for all of her guidance and assistance during the course of this project. To my other committee members, Dr. Jingdong Ye and Dr. Dmitry Kolpashchikov, thank you for taking the time to be a part of this process. Additionally, I would like to acknowledge those who donated one or more body fluids for this project. vi

8 TABLE OF CONTENTS LIST OF FIGURES... ix LIST OF TABLES... xiii LIST OF ACRONYMS/ABBREVIATIONS... xv CHAPTER ONE: INTRODUCTION... 1 CHAPTER TWO: METHODOLOGY... 6 Sample Preparation... 6 Degradation Samples... 6 Standard RNA Extractions... 7 Standard Organic RNA Extraction... 7 RNeasy Micro Kit... 8 Standard Extractions... 9 Standard Organic Extraction... 9 Investigator Kit /RNA Co-Extraction Methods AllPrep /RNA Mini kit ToTALLY RNA Kit/ Back Extraction TRIzol Extraction/ Back Extraction Chaos Buffer/Spin Columns NCFS Co-extraction DNase I Digestion Quantitation of Isolated and RNA c Synthesis Polymerase Chain Reaction Amplification Detection of Amplified Products Post-PCR Purification Optimization of Co-extraction Methods Testing of Optimized Protocols and RNA Stability CHAPTER THREE: RESULTS (CO-EXTRACTION) Evaluation of Co-extraction Methods vii

9 Standard and RNA Extractions Co-extraction Methods Optimization of Extraction Protocols RNeasy Micro Kit AllPrep /RNA Mini Kit NCFS Co-extraction RNA Detection Testing of Optimized Protocols Sensitivity CHAPTER FOUR: RESULTS ( AND RNA STABILITY) Indoor Samples Room Temperature C C Substrates Carpet Denim Environmental Samples Outside Covered and Uncovered Other Conditions CHAPTER FIVE: DISCUSSION /RNA Co-extraction and RNA Stability CHAPTER SIX: CONCLUSION APPENDIX A: FIGURES APPENDIX B: TABLES REFERENCES viii

10 LIST OF FIGURES Figure 1: Evaluation of and RNA Recovery Using the Five Co-extraction Methods and Standard and RNA Extraction Methods Figure 2: RNA Detection Results for Blood and Saliva Samples Extracted Using the Five Coextraction Methods Figure 3: Profiles Obtained from Blood Samples Using the Five Co-extraction Methods. 69 Figure 4: Profiles Obtained from Saliva Samples Using the Five Co-extraction Methods 70 Figure 5: Effect of Lysis Incubation Time and Temperature on RNA Recovery Figure 6: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the AllPrep /RNA Mini Kit Figure 7: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the NCFS Co-extraction Figure 8: RNA Detection Results Using the Optimized Protocols of the NCFS Co-extraction and AllPrep /RNA Mini Extraction Figure 9: Profiles Obtained Using the Optimized NCFS Co-extraction and AllPrep /RNA Mini Protocols Figure 10: Comparison of and RNA Recovery Using the Optimized NCFS Co-extraction and AllPrep Extractions Figure 11: Profiling Success Rate of Optimized Protocols Figure 12: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood (RNA) Figure 13: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva (RNA) Figure 14: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen (RNA) ix

11 Figure 15: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood () Figure 16: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva () Figure 17: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen () Figure 18: and RNA Recovery from Blood Stains (Swab) Exposed to Various Temperatures Figure 19: and RNA Recovery from Saliva Stains (Swab) Exposed to Various Temperatures Figure 20: and RNA Recovery from Semen Stains (Swab) Exposed to Various Temperatures Figure 21: and RNA Recovery from Vaginal Secretion Swabs Exposed to Various Temperatures Figure 22: and RNA Stability in Blood Stains (Swab) Incubated at 37 C and 56 C Figure 23: and RNA Stability in Saliva Stains (Swab) Incubated at 37 C and 56 C Figure 24: and RNA Stability in Semen Stains (Swab) Incubated at 56 C Figure 25: and RNA Stability in Vaginal Secretion Swabs Incubated at 37 C and 56 C.. 91 Figure 26: and RNA Stability in Blood Stains Made on Different Substrates, 37 C Figure 27: and RNA Stability in Blood Stains Made on Different Substrates, 56 C Figure 28: and RNA Profiles (Blood Stains on Different Substrates) Figure 29: and RNA Stability in Saliva Stains Made on Different Substrates, 37 C Figure 30: and RNA Stability in Saliva Stains Made on Different Substrates, 56 C Figure 31: and RNA Profiles (Saliva Stains on Different Substrates) Figure 32: and RNA Stability in Semen Stains Made on Different Substrates, 37 C x

12 Figure 33: and RNA Stability in Semen Stains Made on Different Substrates, 56 C Figure 34: and RNA Profiles (Semen Stains on Different Substrates) Figure 35: Recovery of and RNA from Blood Stains (Outside) Figure 36: Average HBB and ALAS2 Peak Heights: Blood (Outside) Figure 37: Average Allele Peak Heights: Blood (Outside) Figure 38: and RNA Stability in Blood Stains Exposed to Different Environmental Conditions Figure 39: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Covered) Figure 40: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Uncovered) Figure 41: Recovery of and RNA from Saliva Stains (Outside) Figure 42: Average HTN3 Peak Height: Saliva (Outside) Figure 43: Average Allele Peak Heights: Saliva (Outside) Figure 44: and RNA Stability in Saliva Stains Exposed to Different Environmental Conditions Figure 45: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Covered) Figure 46: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Uncovered) Figure 47: Recovery of and RNA from Semen Stains (Outside) Figure 48: Average PRM2 and TGM4 Peak Heights: Semen (Outside) xi

13 Figure 49: Average Allele Peak Heights: Semen (Outside) Figure 50: and RNA Stability in Semen Stains Exposed to Different Environmental Conditions Figure 51: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Covered) Figure 52: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Uncovered) Figure 53: Recovery of and RNA from Vaginal Secretion Swabs (Outside) Figure 54: Average MUC4 Peak Heights: Vaginal Secretion (Outside) Figure 55: Allele Peak Heights: Vaginal Secretions (Outside) Figure 56: and RNA Stability in Vaginal Secretion Swabs Exposed to Different Environmental Conditions Figure 57: and RNA Profiles Obtained from Vaginal Secretion Swabs Exposed to the Environment (Outside Covered) Figure 58: and RNA Profiles Obtained from Vaginal Secretion Swabs Exposed to the Environment (Outside Uncovered) Figure 59: and RNA Recovery from Blood Stains (Shade, Sun, Patio) Figure 60: and RNA Recovery from Saliva Stains (Shade, Sun, Patio) Figure 61: and RNA Recovery from Semen Stains (Shade, Sun, Patio) Figure 62: and RNA Stability in Blood Stains (Shade, Sun, Patio) Figure 63: and RNA Stability in Saliva Stains (Shade, Sun, Patio) Figure 64: and RNA Stability in Semen Stains (Shade, Sun, Patio) xii

14 LIST OF TABLES Table 1: Co-extraction Methods Evaluated Table 2: Body Fluid Markers Table 3: Co-extraction Methods Evaluation: and RNA Detection Results Table 4: RNA Results Obtained Using Optimized and Standard Co-extraction Protocols Table 5: Sensitivity of the Optimized AllPrep and Nucleospin Extractions Using Whole, 1/2, and 1/4 Stains or Swabs Table 6: Sensitivity of the Optimized AllPrep and Nucleospin Co-extraction Methods: Stains 147 Table 7: and RNA Stability in Blood Stains (Swab), 37 C Table 8: and RNA Stability in Blood Stains (Swab), 56 C Table 9: and RNA Stability in Saliva Stains (Swab), 37 C Table 10: and RNA Stability in Saliva Stains (Swab), 56 C Table 11: and RNA Stability in Semen Stains (Swab), 56 C Table 12: and RNA Stability in Vaginal Secretion Swabs, 37 C Table 13: and RNA Stability in Vaginal Secretion Swabs, 56 C Table 14: Substrate s Effect on and RNA Stability in Blood Stains, 37 C Table 15: Substrate s Effect on and RNA Stability in Blood Stains, 56 C Table 16: Substrate s Effect on and RNA Stability in Semen Stains, 56 C Table 17: and RNA Stability in Blood Stains (Outside Covered) Table 18: and RNA Stability in Blood Stains (Outside Uncovered) Table 19: Average Allele Peak Heights (Blood) Table 20: and RNA Stability in Saliva Stains (Outside Covered) xiii

15 Table 21: and RNA Stability in Saliva Stains (Outside Uncovered) Table 22: Average Allele Peak Heights (Saliva) Table 23: and RNA Stability in Semen Stains (Outside Covered) Table 24: and RNA Stability in Semen Stains (Outside Uncovered) Table 25: Average Allele Peak Heights (Semen) Table 26: and RNA Stability in Vaginal Secretion Swabs (Outside Covered) Table 27: and RNA Stability in Vaginal Secretion Swabs (Outside Uncovered) Table 28: Average Allele Peak Heights (Vaginal Secretions) xiv

16 LIST OF ACRONYMS/ABBREVIATIONS ALAS2 aminolevulinate synthase 2 BL blood CE capillary electrophoresis c complementary Ct cycle threshold DEPC diethylpyrocarbonate deoxyribonucleic acid DNase deoxyribonuclease DTT dithiothreitol EDTA ethylenediaminetetraacetic acid HBB hemoglobin-beta chain HTN3 histatin 3 IPC internal positive control VAG1 vaginal secretion primer MMP-10 matrix metalloproteinase-10 mrna messenger RNA MB menstrual blood MUC4 mucin 4 MUC7 mucin 7 NCFS National Center for Forensic Science OS-C outside covered OS-UC outside uncovered PBGD porphobilinogen deaminase PCR polymerase chain reaction P/C/IAA phenol/chloroform/isoamyl alcohol PRM2 protamine 2 RFU relative fluorescence unit RNA ribonucleic acid RNase ribonuclease RT room temperature/reverse transcription RT-PCR reverse transcription-polymerase chain reaction SA saliva SDS sodium dodecyl sulfate SE semen STATH statherin STR short tandem repeat TE tris-edta TGM4 transglutaminase 4 VAG1 vaginal marker VS vaginal secretions xv

17 CHAPTER ONE: INTRODUCTION Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the (deoxyribonucleic acid) found in that material have been well established [1]. STR (short tandem repeat) profiles that are obtained can then be used for making suspect inclusions and exclusions [1]. Initially, evidence gathered during the investigation of a crime is screened for the presence of biological fluids so that potential sources of can be identified [1;2]. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the originated is also of interest as it can provide information about the crime that took place. The conventional serological methods that are used for body fluid identification can be costly, must be performed sequentially, and can consume the often limited amount of available sample, leaving less for analysis [2]. Therefore, there is a need for advances in body fluid identification strategies to reduce the amount of time and sample required to perform both body fluid and analysis. In the past few years, numerous studies have demonstrated the ability to use mrna (messenger ribonucleic acid) expression profiling for the identification of forensic biological stains [2-12]. Cells are differentiated to form various body fluids and tissues, and the cell types contained within those fluids or tissues have a unique set of active genes from which mrna is transcribed, making it possible to identify a fluid or tissue based on the type and abundance of mrna transcripts present [13]. There is not yet widespread use of mrna profiling in forensic casework, but this molecular based approach offers the advantages of improved sensitivity and specificity while making it possible to simultaneously test for multiple body fluids at once [2]. In order for this body fluid typing technique to be utilized in routine forensic casework, RNA of 1

18 sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current analysis methodologies. While several methods that describe the simultaneous isolation of and RNA from the same sample have been reported, few of these studies have performed thorough optimization and validation experiments focused on the often reduced quantity and compromised quality of samples encountered in forensic casework [14-23]. To determine what extraction method would allow for and RNA of high quality to be obtained from forensic samples, several /RNA co-extraction methods were compared (Table 1). The methods evaluated included the AllPrep /RNA Mini kit (Qiagen, Valencia, CA), the ToTALLY RNA kit (Ambion, Inc., Austin, TX), the TRIzol reagent (Invitrogen, Grand Island, NY), an organic based coextraction method developed in-house at the National Center for Forensic Science (NCFS coextraction), and a method which utilizes an in-house chaos buffer and spin columns. The AllPrep /RNA Mini kit is a spin column-based method that allows for the purification of genomic and total RNA consisting of 200 nucleotides (mostly mrna) from animal cells and tissues in a short amount of time [23]. This method was chosen because it is a kit specifically designed for the co-extraction of and RNA and can be automated. Some of the previously described co-extraction methods involve isolating RNA from the aqueous phase and from the organic phase during phenol/chloroform purification which is possible because a lower ph allows for this differential separation of the nucleic acids to occur [15;22;24]. The ToTALLY RNA kit was designed for the isolation of pure RNA by using two separate phenol/chloroform purification steps, but a modified protocol allows for the from the normally discarded organic phases to be isolated using a back extraction buffer [24]. In the same way, the 2

19 TRIzol reagent is normally used for the isolation of RNA from cells and tissues, but protocols are also available for the back extraction of from the organic phase [18;22]. In 2004, an organic /RNA co-extraction method was developed in-house for use with forensic casework samples [14]. Alvarez, et al. showed that this method could be used to successfully isolate and RNA from samples of reduced quality and quantity often encountered in forensic casework; however, it is an organic extraction and requires several hours to complete, so it was not necessarily expected to be the best available co-extraction method. In a recent article, the authors compared several co-extraction methods using fish embryos [22]. They looked at both organic and spin column methods including TRIzol and back extractions, the AllPrep /RNA Micro kit for small samples, and a guanidinium-based chaos buffer used for sample lysis followed by spin column purification. They found that the highest quantity of and RNA obtained without sacrificing quality could be achieved when the chaos buffer was used to lyse the sample and the lysate was then split in half and applied to separate DNeasy and RNeasy Qiagen spin columns. In order to determine which of the above methods would be most suitable for forensic samples, the quantity and quality of and RNA recovered using each of the co-extraction methods were compared. Standard non-co-extraction methods were also performed to establish a basis for comparison. The most promising methods were then chosen for optimization in order to maximize extraction efficiency and nucleic acid recovery. After optimization of a chosen co-extraction protocol, the method could then be used as a means to compare the relative stability of and RNA in the same environmentally compromised biological fluid stain. Messenger RNA has a limited lifetime in the cell and is often 3

20 quickly degraded because of its role as a control for protein synthesis [25-27]. Additionally, in alkaline conditions the 2 OH group of the ribose sugar which deoxyribose does not have makes it more susceptible to hydrolysis resulting in strand cleavage [28]. For these reasons and because of the abundant endogenous and exogenous RNases present that can degrade RNA, it is often assumed that in forensically relevant stains would remain stable for a longer period of time [25;29]. This concern about RNA stability has in some ways prevented the acceptance of mrna profiling in forensic casework [30]. Unfavorable conditions such as UV light, humidity, bacterial growth, and high temperatures have all been shown to accelerate the degradation of both and RNA in dried stains [31-35], but a thorough investigation of the relative degradation rates of and RNA in the same sample has not been performed. Hydrolysis is a major cause of degradation, and it results in base loss and strand scission which can then impede amplification of a target sequence [36-38], but the dehydrated state of a body fluid stain greatly reduces the occurrence of hydrolysis as well as other potentially harmful chemical reactions [34]. in dried stains has been shown to remain stable for years, but even under proper storage conditions some degradation can still occur [31;32]. It was thought that RNA in dried stains would not remain stable for as long, but studies have shown that RNA can also be obtained and analyzed after several years [29;31]. The effect that storage conditions can have on RNA recoverability and stability has also been evaluated, and RNA suitable for body fluid identification by RT-PCR (reverse transcription-polymerase chain reaction) analysis was obtained from body fluid stains stored under various conditions at room temperature after 547 days [33]. 4

21 The studies showing the stability of RNA are promising, but it is important to know if one can expect RNA body fluid markers to be detected in a degraded sample when a profile can still be obtained, if RNA will not be recoverable for as long as, or if RNA can be successfully analyzed beyond the point that a profile can be obtained. In order to determine this relationship, we incubated forensically relevant body fluid stains (blood, saliva, semen, and vaginal secretions) at different set temperatures for increasing lengths of time as well as outdoors to determine the relative effects of environmental conditions (i.e. sunlight, rain, humidity) on and RNA stability. Stains were also made on different substrates that biological fluid stains could be found on to determine if the substrate itself affects the relative level of and RNA degradation. 5

22 CHAPTER TWO: METHODOLOGY Sample Preparation Body fluids were collected from volunteers using procedures approved by the university s institutional review board. Blood was collected by venipuncture, and 50 µl aliquots were deposited onto sterile cotton cloth (t-shirt). For sensitivity testing of the optimized coextraction protocols, 10 µl, 5 µl, 1 µl, and 0.2 µl blood stains were prepared on cotton. Saliva was obtained by swabbing the inside of the cheek with a cotton-tipped swab. For sensitivity testing, liquid saliva was collected in sterile 15 ml centrifuge tubes and 50 µl, 25 µl, 10 µl, 5 µl, and 1 µl stains were then made on sterile cotton cloth. Semen was collected in 50 ml centrifuge tubes, and swabs of the sample were taken or 50 µl, 25 µl, 10 µl, 5 µl, and 1 µl stains were made for sensitivity testing. Semen-free vaginal secretion and menstrual blood swabs were collected by swabbing the vaginal cavity with sterile cotton-tipped swabs. During optimization and sensitivity testing, ½ and ¼ buccal, semen, vaginal, and menstrual blood swabs and ½ and ¼ of 50 µl blood stains were also extracted. Stains or swabs were allowed to air dry at room temperature and were then stored at -20 C until needed. Degradation Samples For the comparison of and RNA degradation rates, body fluid stains or swabs were made for blood, saliva, semen, and vaginal secretions. The body fluids were obtained from the donors as described above except that non-edta tubes were used to collect blood. The substrates upon which stains were made included t-shirt cotton (100% cotton, white), denim 6

23 (100% cotton, medium blue), carpet (nylon, tan, short fibers; used), and swabs (sterile cotton tipped applicators). Stains/swabs prepared for three donors per body fluid were then incubated protected from light at room temperature (~22ºC, 53% humidity), 37ºC (incubator), or 56ºC (3 incubators to accommodate all samples) with the ambient humidity of the room/incubator for 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, and 1 year, and Time 0 control samples were also prepared. Blood, saliva, semen, and vaginal secretion samples (previously prepared) from four (blood and saliva) or two donors (semen and vaginal secretions) were also incubated outside exposed to light, heat (average high of 36 C), humidity, and rain (uncovered) or light, heat, and humidity (covered) for 1 day, 3 days, 1 week, 4 weeks, 3 months, 6 months, and 1 year. A few additional blood, saliva, and semen stains (1 donor per fluid, previously prepared) were incubated in the shade, sun, and on a patio for various lengths of time. Standard RNA Extractions To establish what results would be expected using standard, non-co-extraction methods, both organic and spin column-based RNA extractions were completed using 50 μl dried blood stains and saliva, semen, vaginal secretion, and menstrual blood swabs. Extractions were performed twice to verify that results could be duplicated. Standard Organic RNA Extraction A standard organic RNA extraction using guanidine isothiocyanate-phenol:chloroform was performed [39]. A mixture of 500 µl denaturing solution (guanidine isothiocyanate, 0.02 M 7

24 sodium citrate dihydrate, 0.5% sarkosyl) and 3.6 µl of β-mercaptoethanol was preheated for 10 minutes at 56 C. The sample (stain or swab) was placed into a safe-lock microcentrifuge tube (Eppendorf, Hauppauge, NY) and incubated in the denaturing solution mixture for 30 minutes at 56 C. The swab or cloth pieces were placed into a spin basket (Promega, Madison, WI) which was then inserted back into the extraction tube, and the sample was centrifuged at 8,160 x g for 10 minutes. The spin basket and its contents were discarded. Fifty microliters of 2 M sodium acetate and 600 µl of acid-phenol:chloroform 5:1 (ph 4.5, Ambion, Inc.) were added to the lysate to create a phase separation, maintaining the RNA in the aqueous phase. The sample was incubated at 4 C for 30 minutes and then centrifuged at full speed (16,000 x g) for 20 minutes to separate the phases. The aqueous phase was transferred to a new 1.5 ml microcentrifuge tube, and the RNA was precipitated at -20 C for at least 1 hour in a mixture of 500 µl isopropanol and 2 µl GlycoBlue glycogen carrier (Ambion, Inc.). The sample was centrifuged at full speed for 20 minutes, and the resulting pellet was washed with 75% ethanol/25% DEPC-treated water, dried in a vacuum centrifuge for 5 minutes, and incubated at 60 C in 12 µl of RNA Secure Resuspension Solution (Ambion, Inc.) for 10 minutes to resolubilize the pellet. RNeasy Micro Kit The RNeasy Micro kit (Qiagen) was used as the standard spin column method. The extraction was completed either by hand or using the QIAcube robot from Qiagen following the protocol provided by the manufacturer. The sample was first lysed in the provided Buffer RLT containing β-mercaptoethanol. One volume of 70% ethanol was added to the lysate, and the entire sample was transferred to the RNeasy MinElute spin column. The column was washed 8

25 with Buffer RW1, and an on-column DNase I digestion was then performed. A mixture of 10 µl DNase I stock and 70 µl Buffer RDD was applied to the column which was then incubated at room temperature for 15 minutes. Buffers RWI and RPE and 80% ethanol were used to wash the column, and the RNA was eluted into a new 1.5 ml tube with 14 µl of RNase-free water (provided). Standard Extractions As with the RNA extractions, two different standard, non-co-extraction methods, both organic and spin column-based, were used. Standard Organic Extraction A standard organic extraction, as previously described, was performed [40]. The sample was incubated at 56 C overnight in 400 µl of stain extraction buffer (10 mm Tris-HCl (ph 8.0), 0.1 M NaCl, 0.5 M EDTA, 20% SDS), 20 µg/µl proteinase K, and µg/µl dithiothreitol (DTT, semen samples only). The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. Four hundred microliters of phenol/chloroform/isoamyl alcohol (P/C/IAA, 25:24:1, ph 6.6) was added to create a separation of the organic material and. The sample was centrifuged at full speed to separate the layers, and the aqueous phase was removed to a new 1.5 ml tube. was precipitated at -20 C for at least 1 hour using cold 100% ethanol. The sample was centrifuged at full speed for 15 minutes to pellet the, and the pellet was washed 9

26 with 70% ethanol, dried in a vacuum centrifuge for 5 minutes, and then resolubilized in 100 μl of TE -4 (10 mm Tris, 0.1 mm EDTA, ph 7.5) at 56ºC overnight. Investigator Kit The QIAamp Investigator kit (Qiagen) was chosen as the standard spin-column extraction method, and it was performed following the manufacturer s instructions. The provided Buffer ATL and proteinase K were added to the sample which was then incubated at 56ºC for 1 hour. The sample was vortexed every 10 minutes to facilitate lysis. Buffer AL was added, and the sample was incubated at 70ºC an additional 10 minutes for complete lysis. Ethanol (100%) was added to the lysate to create proper binding conditions, and the sample was transferred to the QIAamp MinElute spin column which selectively binds. Contaminants were washed from the column using the provided Buffers AW1 and AW2 and 70% ethanol. The column was incubated at room temperature for 10 minutes to dry the membrane, and the was then eluted in 50 µl of Buffer ATE. /RNA Co-Extraction Methods Each of the co-extraction methods evaluated (Table 1) was performed twice using a 50 µl blood stain (two different donors) and a buccal swab (the same donor). AllPrep /RNA Mini kit The AllPrep /RNA Mini kit extraction (Qiagen) was performed either manually or using the QIAcube robot following the manufacturer s instructions. The sample was lysed 10

27 briefly using 350 µl of the provided Buffer RLT and a 0.01 volume of β-mercaptoethanol. The lysate was applied to the AllPrep Mini spin column, and the flow-through was then mixed with 70% ethanol to create proper conditions for the selective binding of RNA to the RNeasy spin column. Contaminants were washed from the column using the Buffers AW1 and AW2 and from the RNA column using the Buffers RW1 and RPE. The was eluted in 100 µl of Buffer EB, and RNA was eluted in 30 µl of RNase-free water. ToTALLY RNA Kit/ Back Extraction The ToTALLY RNA kit (Ambion, Inc.) was designed for RNA extractions, but the protocol can be adapted to allow for isolation as well [24]. The manufacturer s protocol was followed with a few adjustments. The sample was lysed in 300 µl of the provided guanidinium based Denaturation Solution for minutes at room temperature. The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. The RNA was then purified by first using 1 volume of P/C/IAA (ph 6.6). The sample was vortexed for 1 minute, and then incubated on ice for 5 minutes. After centrifugation for 5 minutes at 8,160 x g, the aqueous phase was transferred to a new tube, and the organic phase was set aside. Sodium acetate (1/10 volume) and acid-phenol:chloroform (ph 4.5, 1 volume) were added to the aqueous phase, and the same steps were followed as with the P/C/IAA purification. The aqueous phase was transferred to a new tube, and the organic phase was set aside. The RNA was precipitated from the aqueous phases using an equal volume of isopropanol at -20 C for at least 30 minutes, and the resulting pellet was washed with 70% ethanol and then resuspended in 12 µl of RNA Secure 11

28 Resuspension Solution as used with the standard RNA organic extraction. The organic phase from both the P/C/IAA and acid-phenol:chloroform purifications were combined, and a back extraction buffer (0.1 M NaCl, 10 mm Tris-HCl, ph 8.0, 1 mm EDTA, 1% SDS, ph 12) was added to isolate the remaining in the organic phases. The sample was incubated at -20 C for 10 minutes and centrifuged at 8,160 x g for 20 minutes. The aqueous layer was transferred to a new 1.5 ml tube, and the was precipitated with 2 volumes of cold 100% ethanol at -20 C for at least 1 hour. The pellet was washed with 70% ethanol and then resuspended in 50 µl TE -4 for 5 minutes at 56 C. TRIzol Extraction/ Back Extraction The TRIzol LS reagent (Invitrogen) is used for RNA extractions, but can also be isolated by adding a back extraction buffer to the saved organic phase [18;22]. Eight hundred microliters of the TRIzol reagent which contains both guanidine isothiocyanate and phenol was used to lyse the sample, and the cut sample pieces were then placed into a spin basket which was inserted back into the extraction tube and centrifuged at full speed for 5 minutes. Two hundred microliters of chloroform was added to create a phase separation, and the sample was centrifuged at 8,160 x g for 15 minutes. The aqueous phase was transferred to a new 1.5 ml tube, and the organic phase was set aside. The RNA was precipitated with 500 µl isopropanol and 2 µl GlycoBlue glycogen carrier at room temperature for 10 minutes, and the sample was then centrifuged at full speed for 10 minutes. The resulting pellet was washed with 1 ml of 75% ETOH/25% DEPC water, air dried for 10 minutes, and resuspended in 12 µl of RNA Secure Resuspension Solution. A back extraction buffer (4 M guanidine thiocyanate; 50 mm sodium 12

29 citrate; 1 M Tris, ph 8.0) was added to the saved organic phase to isolate the remaining. The sample was incubated at room temperature for 10 minutes and centrifuged at full speed for 15 minutes. The aqueous phase was transferred to a new 1.5 ml tube, and the was precipitated at -20 C for 1 hour in an equal volume of isopropanol. The pellet was washed with 70% ethanol, dried in a vacuum centrifuge, and then resuspended in 50 µl TE -4 at 56 C for 10 minutes. Chaos Buffer/Spin Columns In an article by Triant and Whitehead (2008), various /RNA co-extraction methods were tested, and their results showed that using a Chaos Buffer (4.5 M guanidine isothiocyanate, 2% sarkosyl, 50 mm EDTA, 0.1 M β-mercaptoethanol) and spin columns allowed them to isolate and RNA of high quality and quantity from the same sample [22]. Four hundred microliters of the chaos buffer was added to the sample which was incubated at 56 C for 30 minutes, and the lysate was then split in half, one portion (~200 µl) being applied to an RNeasy spin column and the other being applied to a DNeasy Blood and Tissue spin column (Qiagen). The manufacturer s protocols were followed for the RNeasy Mini and DNeasy Blood and Tissue kits. The RNA was bound to the RNeasy column, contaminants were washed from the column with the Buffers RW1 and RPE, and the RNA was then eluted in 30 µl of RNase-free water. The portion was mixed with Buffer AL and 100% ethanol and then applied to the DNeasy column, the column was washed with the Buffers AW1 and AW2, and the was eluted in 100 µl of Buffer AE. 13

30 NCFS Co-extraction The in-house NCFS co-extraction method used was developed by Alvarez, et al. in 2004 [14]. The sample was first lysed in 500 µl of the same stain extraction buffer used with the standard organic extraction and proteinase K. DTT (10% of extraction volume) was added to semen samples only. The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. Fifty microliters of sodium acetate (2 M) and 600 µl of acid-phenol:chloroform (ph 4.5) were added to the sample to create a phase separation. The sample was incubated at 4 C for 20 minutes and then centrifuged at full speed for 20 minutes. The aqueous phase (~500 µl) was transferred to a new 1.5 ml tube labeled, and half of it was then transferred to another tube labeled RNA. and RNA were then precipitated following the same procedures used with the standard organic and RNA extractions. was precipitated in ethanol and then resolubilized in 50 μl TE -4 at 56 C for 45 minutes, while RNA was precipitated in isopropanol and resolubilized in 12 μl of RNA Secure Resuspension Solution. DNase I Digestion All RNA extracts were treated with DNase to remove any contaminating that might have remained in the sample. Six units of TURBO DNase I (2 U/µL, Ambion, Inc.) and 10X DNase Buffer were added, and the sample was incubated at 37 C for 1 hour. The enzyme was inactivated by incubating the sample at 75 C for 10 minutes [41-43]. A slightly different DNase digestion method was used for the degradation study: TURBO -free DNase (Ambion) and 0.1 volume of 10X TURBO DNase buffer were added to the RNA extract, and the sample 14

31 was then incubated at 37 C for 20 minutes. To inactivate the enzyme, a 0.1 volume of DNase Inactivation Reagent was added. The samples were stored at -20 C until needed. Quantitation of Isolated and RNA The Quantifiler Human Quantification kit (Applied Biosystems, Foster City, CA) and the ABI 7500 Fast Real Time PCR system were used for quantitation. Fast 96-well plates were prepared with 0.8 µl of standard or sample and 9.2 µl of PCR reaction/primer mix. The cycling conditions were 10 minutes at 95ºC followed by 45 cycles of 10 sec at 95ºC and 1 minute at 60ºC. concentrations were calculated based on Ct values of the quantitation standards. The Quant-iT Ribogreen assay (Molecular Probes, Eugene, OR) was used for RNA quantitation using the high-range standard concentrations (20 ng/ml to 1 µg/ml) [44]. The 20X TE buffer provided with the kit was diluted to 1X and mixed with 2 µl of sample in a 96-well plate. The Ribogreen reagent was diluted in the 1X TE and 100 µl of the solution was added to the sample wells for a total assay volume of 200 µl. The plate was incubated in the dark for 3 minutes, and the fluorescence emission at 535 nm (485 nm excitation) was measured using a Wallac Victor 2 microplate reader (Perkin Elmer Life Sciences, Boston, MA). Total RNA concentrations of the samples were calculated based on a standard curve created using the fluorescence measurements of the quantitation standards. 15

32 c Synthesis Reverse transcription (RT) was used to synthesize complementary (c) strands from the isolated mrna transcripts [45;46]. Three different RT reactions were used over the course of this project. The reactions performed included using reagents from Ambion (10X firststrand buffer, random decamer primers, 10 mm dntp mix (Applied Biosystems), SUPERase- In RNase Inhibitor, and Moloney Murine Leukemia Virus-Reverse Transcriptase), the SuperScript III First-Strand Synthesis System (Invitrogen), and the High-Capacity c Reverse Transcription kit (Applied Biosystems). The target amount of RNA used in the reaction was 50 ng. For samples which contained less than 50 ng, the entire extract was used. For the first RT reaction listed, sample extracts were preheated at 75 C for 3 minutes, the RT reagents mix was added for a total volume of 30 µl, and the samples were then incubated for 1 hour at 42 C and for 10 minutes at 95 C to inactivate the enzyme. For the SuperScript III RT reaction, samples were preheated with dntp mix and random primers at 65 C for 5 minutes, the RT mix (0.1 M DTT, 25 mm MgCl 2, 10X RT buffer, RNaseOUT RNase inhibitor, and SuperScript III RT) was added for a total volume of 20 µl, and the samples were incubated at 25 C for 10 minutes, at 50 C for 50 minutes, and at 85 C for 5 minutes to inactivate the enzyme. For the ABI High Capacity RT reaction, sample extracts were preheated at 75 C for 3 minutes, the RT mix (10X RT buffer, 100 mm dntp mix, 10X random primers, and MultiScribe RT) was added for a total volume of 20 µl, and the samples were incubated at 25 C for 10 minutes, at 37 C for 120 minutes, and at 85 C for 5 minutes to inactivate the enzyme. An RT+ reagent blank containing the RT reagents and water and RT- controls containing sample extract but no reverse transcriptase enzyme were included with each reaction. The RT- controls were used to show that 16

33 no or pseudogenes were being amplified and that only peaks from true mrna transcripts were observed. The first RT method (Ambion) was used during the co-extraction methods evaluation and the Superscript III First-Strand kit was used with the optimized methods. For the degradation study, only the ABI High Capacity RT kit was used. Polymerase Chain Reaction Amplification For all of the co-extraction evaluation and optimization samples, the AmpFlSTR SGM Plus PCR Amplification Kit (Applied Biosystems) was used for STR amplification [47]. The target amount of used in the reaction was 1-2 ng or pg for a half reaction. The cycling conditions were 11 minutes at 95 C, followed by 28 cycles of 1 minute at 94 C, 1 minute at 59 C, and 1 minute 72 C, and a final extension of 45 minutes at 60 C. The SGM Plus kit amplifies 10 STR loci plus Amelogenin for sex determination with 2.5 U/1.25 U AmpliTaq Gold Polymerase (5 U/µL, Applied Biosystems) in a 25 µl reaction or 12.5 µl half reaction volume. For the samples of the degradation study, the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems) was used [48]. The target amount of was the same as used with the SGM Plus kit. The Identifiler kit amplifies 15 STR loci plus Amelogenin. The cycling conditions used were also the same except that the final extension was 60 minutes at 60 C. For RNA body fluid testing, 3 µl of c product was amplified using an mrna body fluid typing multiplex. A mix of 10X PCR reaction buffer (Applied Biosystems), 10 mm dntps (Applied Biosystems), 25 mm MgCl 2 (Applied Biosystems), and 1.25 or 1.5 U AmpliTaq Gold was prepared and a combination of the following primers (Applied Biosystems, Invitrogen) was 17

34 added: PBGD or ALAS2 for blood, HTN3 or STATH for saliva, PRM2 for semen, MUC4 and/or VAG1 (unpublished) for vaginal secretion, and MMP-10 for menstrual blood. Table 2 lists the primer sequences of the body fluid markers used. The primer sequences of PBGD, PRM2, and MUC4 were obtained from published sources [9]. Different PRM2 primers (F: 5 -VIC- GGCGCAAAAGACGCTCC, R: 5 -GCCCAGGAAGCTTAGTGCC) were used during optimization to amplify smaller c fragments for better amplification efficiency [10]. The sequences and sizes for the HTN3, STATH, ALAS2, and MMP-10 markers differed from published sequences and were designed using Primer3 Online primer design software. The cycling conditions were 11 minutes at 95 C, 35 cycles of 94 C for 20 sec, 58 C for 30 sec, and 72 C for 40 sec, and a final extension of 5 minutes at 72 C. During the optimization of the coextraction methods, the multiplex used employed different primers and therefore, slightly different cycling conditions: 11 minutes at 95ºC, 35 cycles of 94ºC for 20 sec, 60ºC for 30 sec, and 72ºC for 40 sec, and a final extension of 80 minutes at 72ºC. For the degradation samples, we wanted to maximize the possibility of RNA being detected (i.e. amplification with highest sensitivity) so singleplexes or duplexes were used instead of a multiplex. HBB [8] and ALAS2 singleplexes were used for blood, an HTN3 singleplex was used for saliva, a PRM2 and TGM4 [6] duplex was used for semen to account for the possibility of a vasectomized donor, and a MUC4 singleplex (FAM labeled) was used for vaginal secretions. The same amplification conditions as used with the multiplex (during optimization) were used for the singleplex/duplex reactions except for the HBB singleplex: 11 minutes at 95ºC, 30 cycles of 94ºC for 20 sec, 57ºC for 30 sec C per cycle, and 72ºC for 40 sec, and a final extension of 30 minutes at 72ºC. 18

35 Detection of Amplified Products One microliter or 1.5 µl of amplified RNA and products was denatured in Hi-Di formamide (Applied Biosystems) containing either the GeneScan 500 LIZ or ROX size standard (Applied Biosystems). Samples were analyzed by capillary electrophoresis (CE) using either the ABI 3130 or 310 Genetic Analyzer, and results were analyzed using GeneMapper and GeneScan software, respectively. Post-PCR Purification For some RT-PCR amplification reactions, the presence of dye blobs interfered with interpretation of results, so the MinElute PCR Purification kit (Qiagen) was used to clean-up amplified samples using the QIAcube robot. The MinElute kit was also used to increase peak heights in other instances. The entire volume (25 µl) of PCR product was combined with 75 µl Buffer EB, and then 5 volumes of Buffer PB were added. The sample was applied to the MinElute column, Buffer PE was added, and the purified c was then eluted in 15 µl of Buffer EB. One microliter of sample was still used for CE analysis. Optimization of Co-extraction Methods An organic (NCFS co-extraction) and spin column (AllPrep /RNA Mini extraction) extraction method were chosen for optimization based on the initial evaluation of the coextraction methods. Due to low RNA yields obtained with the spin column extractions, attempts were first made to increase the amount of RNA obtained using the standard RNeasy Micro kit 19

36 extraction. The extraction was performed using various lysis incubation times (1 hour, 3 hours, overnight) and temperatures (room temperature, 37ºC, and 56ºC) with duplicate 50 µl blood stains on cotton (whole stain used) and buccal swabs (whole swab used). Additional extractions were also completed using carrier RNA provided with the kit, ½ and ¼ of 50 µl blood stains to determine if the column was being clogged when using a whole stain, and the lysis step used for the standard organic RNA extraction (Denaturing solution, β-mercaptoethanol; 30 minutes at 56ºC). The RNeasy Micro kit extraction was then repeated with duplicate semen, vaginal secretion, and menstrual blood swab samples using the standard conditions and the altered conditions which resulted in the most RNA obtained from the blood and saliva samples. Optimization of the AllPrep /RNA Mini kit extraction began with using the same lysis incubation times (1 hour, 3 hours, overnight) and temperatures (RT, 37ºC, and 56ºC) that were used with the RNeasy Micro kit. These extractions were first performed with blood and saliva samples only, and then later the standard conditions (no incubation) and the altered conditions which resulted in improved and RNA recovery were used with semen, vaginal secretions, and menstrual blood swab samples. Further attempts to optimize the AllPrep protocol included substituting the RNeasy MinElute columns provided with the RNeasy Micro kit for the RNeasy Mini columns normally used with the extraction, substituting DTT for β- mercaptoethanol, adding carrier RNA, and omitting β-mercaptoethanol. Attempts were then made to optimize the NCFS co-extraction. To decrease the time required to perform the extraction and reduce the presence of PCR inhibitors, Nucleospin Clean-up XS and Nucleospin RNA Clean-up XS columns (Macherey-Nagel, Bethlehem, PA) were used to replace the precipitation step of the NCFS co-extraction. The aqueous phase was 20

37 split in half as normal, but then an equal volume (~250 µl) of Buffer RCU was added to the RNA fraction, and the sample was then applied to the Nucleospin RNA column while the fraction was adjusted to 800 µl with TE -4 and 200 µl NT binding buffer was added before then applying the sample to the Nucleospin column [49]. Centrifugation steps were carried out at 11,000 x g. The RNA and columns were washed with the provided Buffer RA3 and B5, respectively. RNA was then eluted in 10 µl of RNase-free H 2 O, and was eluted in 20 µl of Buffer BE. The NCFS co-extraction was also performed using the standard RNA lysis step (Denaturing solution, β-mercaptoethanol; 30 minutes at 56ºC) with and without Nucleospin columns and substituting P/C/IAA for acid-phenol:chloroform. The lysis incubation length was also varied using 15 minute, 30 minute, and 3 hour incubations at 56 C. Testing of Optimized Protocols Larger sample sets were used to test the optimized co-extraction protocols, and the sensitivity of the methods was also evaluated. Samples from a total of 16 donors were used for blood and ½ and ¼ stains were also extracted for 10 of these donors. For saliva, buccal swabs from 10 donors were used, for semen and vaginal secretion, swabs from 8 donors were used, and for menstrual blood, swabs from 5 donors were used. In some cases additional testing was necessary to obtain a positive RNA result (different RT method, altered PCR cycling conditions, RNA singleplex, etc.), and for any RNA samples that were still negative after additional testing, a new swab or stain from that donor was extracted. 21

38 and RNA Stability The NCFS co-extraction with Nucleospin columns was used for the extraction of the samples prepared for the /RNA degradation study. As mentioned previously, the Identifiler kit was used for STR typing and a duplex or singleplex was used for mrna body fluid typing. A standard RNA extraction using Nucleospin columns was also performed with pristine blood, saliva, semen, and vaginal secretion samples to provide non-co-extraction controls to amplify with the degraded samples. 22

39 CHAPTER THREE: RESULTS (CO-EXTRACTION) Evaluation of Co-extraction Methods Standard and RNA Extractions Standard non-co-extractions, both organic and spin column-based, were performed using blood and saliva stains to provide a basis for comparison for the co-extraction methods. For the standard extractions, on average about 4.5 times and 2 times more was recovered using the organic extraction method as compared to the spin column method for blood and saliva, respectively (Figures 1A and 1B). Both organic and spin column extractions produced enough product for downstream reactions, and full STR profiles (SGM Plus ) could be obtained upon CE analysis. For the standard RNA extractions, on average about 8.5 times more RNA was obtained from the blood samples and 18.5 times more was obtained from saliva using the organic extraction compared to the spin column method (Figures 1A and 1B). The RNeasy Micro kit protocol is not adapted for use with forensic samples like the Investigator kit which may explain why the difference in yield between the organic and spin column methods was so much greater for RNA. Additionally, only RNA >200 bases is isolated using spin columns, so the extracts would not contain small RNAs that would be isolated with an organic extraction. Despite the low yields, the HTN3 saliva marker was still detected in the saliva samples from each extraction, and either the PBGD or ALAS2 blood marker was detected in the blood samples. 23

40 Co-extraction Methods Several /RNA co-extraction methods (Table 1) were evaluated using a 50 µl blood stain and a buccal swab (extractions performed twice) to determine which method would be the most suitable for use with forensic samples. A comparison of the co-extraction methods and the standard and RNA extraction methods was made based on the quantity of nucleic acids recovered, the ability to obtain complete STR profiles, and the ability to identify the body fluid origin of the sample using an RNA multiplex. Figure 1 shows a comparison of the average amount of and RNA recovered from the blood (Figure 1A) and saliva (Figure 1B) stains using the different standard and co-extraction methods. For the co-extraction methods evaluated, on average the most and RNA was recovered (without sacrificing one for the other) when using the NCFS co-extraction method. About half as much was recovered compared to the standard organic extraction, but there were still several hundred nanograms of available which is more than enough for analysis. The amount of RNA recovered from the blood and saliva samples was comparable to the standard organic extraction. Table 3 summarizes the and RNA detection results for the five co-extraction methods and the standard non-co-extractions. Full autosomal STR profiles were obtained for all samples using each of the extraction methods except for one of the blood samples extracted with the NCFS co-extraction which was likely due to the presence of PCR inhibitors (heme compound) as later observed (see Testing of Optimized Protocols). The two co-extractions, ToTALLY RNA and TRIzol, which utilize back extraction of from the organic phase, produced adequate RNA yields, but there were issues with obtaining RNA profiles for the blood 24

41 samples which, again, could have been caused by insufficient removal of the heme compound (hematin) [50]. Relatively low yields were also obtained (compared to the other methods), but there was still enough available to produce full STR profiles. The two methods that utilize spin columns, the AllPrep /RNA Mini kit and Chaos buffer/spin column extraction, produced comparatively better yields but relatively low RNA yields with generally insufficient amounts to input 50 ng into the RT reaction. For the blood samples, PBGD was not initially detected, but the samples were amplified again using the more sensitive ALAS2 primers, and ALAS2 was then detected in one of the two blood samples for each extraction. HTN3 or STATH was detected in both of the saliva samples extracted using all five co-extraction methods. Examples of the electropherograms obtained for the five co-extraction methods are shown in Figure 2 (RNA), Figure 3 (-blood), and Figure 4 (-saliva). Based on the results obtained from the standard non-co-extraction methods and the five co-extraction methods, the AllPrep /RNA Mini kit (spin column) and NCFS co-extraction (organic) were chosen for further optimization. Optimization of Extraction Protocols RNeasy Micro Kit Due to the low RNA yields obtained with the spin column extraction methods compared to the organic methods, several optimization experiments were first performed with the RNeasy Micro kit. Duplicate blood and saliva samples were incubated in the provided lysis 25

42 buffer for various lengths of time and at different temperatures to determine if incomplete lysis could be the cause of the low yields. The average RNA yields for the different conditions and times are shown on Figure 5. None of the extractions using the various incubation times and temperatures produced enough RNA to input 50 ng into a RT reaction, but RNA markers were still detected except for the blood samples extracted using the overnight incubation at 56ºC which may have caused some degradation of the RNA. Incubation at room temperature or 37ºC did not seem to have a significant effect on RNA yields. The highest RNA yield that could be obtained using the lysis buffer provided with the kit (Buffer RLT) was achieved by incubating the samples at 56ºC for 1 hour with total yields around 70 ng for blood (compared to ~33 ng) and 60 ng for saliva (compared to ~17 ng) (Figures 5A and 5B). However, when the lysis step normally used for the standard organic RNA extraction (Denaturing solution, β-mercaptoethanol and incubation at 56ºC for 30 minutes) was used, slightly more RNA was obtained from the blood samples, and over two times more was obtained from the saliva samples (data not shown) indicating that by altering the lysis step, RNA yields could be improved. Extractions were then performed with semen, vaginal secretion, and menstrual blood samples using the standard conditions, the 1 hour lysis incubation at 56ºC, and the organic extraction lysis step. An increase in RNA obtained from semen (~193 vs. 259 ng; 357 ng) and vaginal secretion (~126 vs ng; 760 ng) samples was observed for both of the modified lysis steps; however, there appeared to be clogging of the spin column with the vaginal secretion samples as not all of the liquid was flowing through the column in one centrifugation step. The RNA yield from the menstrual blood samples did not seem to be affected greatly by altering the lysis step (~61 vs. 43 ng; 67 ng). The PRM2, MUC4, and MMP-10 markers were detected in all 26

43 of the semen, vaginal secretion, and menstrual blood samples, respectively, when using the modified protocols. AllPrep /RNA Mini Kit Since RNA yields were, for the most part, increased by altering the lysis step of the RNeasy Micro extraction, the AllPrep kit extractions were also performed with duplicate blood and saliva samples using the same incubation times and temperatures in an attempt to increase yield. Generally low RNA concentrations (<1 ng/µl) were obtained except for when the samples were incubated at 56ºC prior to being transferred to the spin column. Also, ALAS2 was only detected in both of the blood samples when an incubation of 1 hour or 3 hours at 56 C was used. HTN3 was detected in almost all of the saliva samples despite the low RNA amounts available for analysis. Sufficient quantities of were recovered for analysis from all of the samples, and full STR profiles were obtained. Based on the total yields (shown in Figure 6), incubation for 3 hours at 56ºC appeared to allow for the greatest amount of RNA (blood: 80 ng, saliva: 262 ng) and (blood: 615 ng, saliva: 3360 ng) to be recovered, so extractions were then performed with duplicate semen, vaginal secretion, and menstrual blood samples using these conditions. There did not appear to be an increase in RNA for the semen (~320 vs. 306 ng) or menstrual blood (~285 vs. 116 ng) samples, and the RNA yields for the vaginal secretion samples were still considerably low (~12.5 vs. 73 ng) compared to how much RNA was obtained with the RNeasy Micro kit (Figure 6B). PRM2 and MMP-10 were detected in the semen and menstrual blood samples extracted with the modified lysis step, but MUC4 was not detected in the vaginal secretion samples. yields 27

44 were improved, however, by incubating the samples at 56ºC for 3 hours (semen: ~2075 vs ng; vaginal secretion: ~4880 vs ng; menstrual blood: ~1240 vs ng) (Figure 6A). Other attempts were then made to determine if and RNA yields could be improved any further. These modifications included substituting RNeasy MinElute columns provided with the RNeasy Micro kit for the RNeasy Mini columns in the AllPrep kit, substituting DTT for β-mercaptoethanol, adding carrier RNA, and omitting β-mercaptoethanol, but none of these changes caused any significant increases in yield and in some cases, caused it to decrease. Due to the conspicuously low RNA yields obtained from the vaginal secretion samples with the AllPrep kit, a few additional attempts were made to improve RNA recovery. First, ½ and ¼ swabs were extracted to determine if the columns were becoming clogged when using a whole swab as had happened with the RNeasy Micro kit columns. However, the RNA yields from these samples were lower than that obtained with a whole swab, and MUC4 was still not detected. The possibility that RNA could be binding to the column and not flowing through to be applied to the RNA column was then considered. To test this hypothesis, a fraction (30 µl) of some of the extracts was treated with DNase, and then processed as a normal RNA sample (Ribogreen quantitation, c synthesis, and RNA multiplex performed). A vaginal secretion RNA marker was in fact detected in one of the DNase treated samples from the extraction using standard conditions and in two of the samples from the extraction using the 3 hour incubation at 56 C, and Ribogreen quantitation revealed that there was around 8 times the amount of RNA in the fraction (~570 ng) than in the actual RNA fraction. extracts from the other body fluids were then tested to see if there was RNA in those samples as well. RNA quantities were not as significant compared to the vaginal secretion samples, but ALAS2, 28

45 HTN3, and PRM2 were still detected in the blood, saliva, and semen fractions from the extraction using incubation at 56 C for 3 hours. Additionally, the DNase treated vaginal secretion samples were amplified using the SGM Plus kit with the maximum amount of sample to determine if was contributing to the RNA quantitation values since Ribogreen also binds to [39], but a partial or no profile was obtained indicating only a small amount of present that would not significantly affect the quantitation results. NCFS Co-extraction A few modifications to the NCFS co-extraction protocol were made to determine if the time to perform the extraction could be shortened and if yields could be increased. Of the alterations made to the NCFS co-extraction, using the Macherey-Nagel Nucleospin columns in place of the precipitation step seemed to provide the greatest improvement to the protocol. By using the Nucleospin Clean-up XS and Nucleospin RNA Clean-up XS columns, the time needed to perform the extraction was reduced by approximately an hour, and any PCR inhibitors (phenol, hematin, etc.) appeared to be removed. For some body fluids, yields were decreased when using Nucleospin columns, but there was still more than sufficient amounts for analysis, and yields from the saliva and semen stains were increased (Figure 7A). The RNA yields were also decreased for the blood and menstrual blood samples, but RNA detection was improved for these samples indicating higher quality product. RNA yields from the saliva and vaginal secretion samples were increased, and did not change drastically for semen (Figure 7B). 29

46 Other variations made to the protocol included using the lysis step for the standard RNA extraction (Denaturing solution, β-mercaptoethanol; 30 minutes at 56 C) and substituting P/C/IAA for acid-phenol:chloroform. Either an increase in RNA accompanied by a decrease in or a decrease in both and RNA was observed when using the RNA lysis. There was an increase in and RNA observed for most of the body fluids compared to the standard method when P/C/IAA was used, but when the extraction was performed a second time, yields were generally lower, and the RNA markers were detected in fewer samples. The lysis incubation time was also altered, using 15 minutes, 30 minutes, 1 hour, and 3 hours. Even with the 15 minute incubation there were still adequate amounts of and RNA obtained. Yields were variable among the different body fluids for both and RNA, so there was not enough consistency to support altering the incubation time from 1 hour. Using the and RNA Nucleospin columns appeared to be the most promising alteration to the NCFS co-extraction protocol as there was still enough product to produce results for all of the and RNA samples (Table 4). RNA Detection A few alterations were made to the RNA multiplex during optimization because there were issues encountered with RNA marker detection (even with the standard extractions), especially for the vaginal secretion samples. Two vaginal markers were included in the multiplex to increase the chance of detection, and the smaller PRM2 semen primers were used to increase sensitivity. The MinElute PCR Clean-up kit was also used to purify all of the c samples amplified using this multiplex to remove any dye blobs and increase signal intensity. 30

47 Additionally, the SuperScript III RT kit was used as it appeared to be a more efficient RT method. Testing of Optimized Protocols When using the optimized NCFS co-extraction and AllPrep protocols with the SuperScript III RT reaction and the modified RNA multiplex, better RNA detection results were obtained compared to using the standard protocols (Table 4). Both of the duplicate body fluid samples tested were positive for their respective body fluid markers when using the optimized protocols. Figures 8 and 9 show examples of the and RNA profiles obtained using the optimized protocols. To further test the optimized co-extraction procedures, larger numbers of samples were used. Initially the standard NCFS co-extraction was also tested to determine if using the Nucleospin columns with the method still appeared to be an improvement when using a larger sample set. After performing the standard NCFS co-extraction with blood stains, no STR profiles were obtained for some of the samples. Upon closer examination of the Real Time PCR results, the presence of PCR inhibitors was indicated by an undetermined or elevated (>25) internal positive control (IPC) Ct value for these samples. Nucleospin columns were used to clean up the extracts, and full profiles could then be obtained. It was suspected that hematin which can sometimes co-purify with the from blood stains was the cause of the inhibition [49], so the standard NCFS co-extraction was repeated with reduced sample sizes (½ and ¼ blood stains), but inhibitors were still present in all of these samples but not when the samples were extracted with the NCFS co-extraction using Nucleospin columns or the AllPrep 31

48 extraction. Nucleospin columns were again used to clean-up the extracts, and full profiles were then obtained. After completing the extraction with saliva samples, surprisingly PCR inhibitors were again present. The source of the PCR inhibitors is unknown, but it is suspected that they were being introduced during the extraction procedure (phenol) and subsequently, not being sufficiently removed. Due to the issues with the PCR inhibitors and the extra labor and time required to remove them, it was decided that the standard NCFS co-extraction method would not be tested any further using the remaining body fluid samples. The NCFS co-extraction with Nucleospin columns already has the columns incorporated into the extraction allowing for the removal of any inhibitors, so only it was tested further along with the AllPrep /RNA Mini extraction. Figure 10 shows the average (A) and RNA (B) yields obtained for the various body fluid samples when using the optimized NCFS co-extraction and AllPrep protocols. On average, more was obtained from the semen, menstrual blood, and vaginal secretion samples (~4 times more) with the AllPrep extraction compared to the Nucleospin extraction, and approximately the same amount was obtained from blood and saliva samples. The amount of RNA recovered from the blood and semen samples was also higher (2.5 times more) with the AllPrep extraction but was much lower for the vaginal secretion samples (7 times less) compared to the Nucleospin extraction, and RNA yields were similar for the saliva and menstrual blood samples. Full autosomal STR profiles were obtained for all of the samples of each body fluid using both extraction methods. Figure 11 shows the and RNA profiling success rate of the two extraction methods. ALAS2 was detected in all 16 of the blood RNA samples using the AllPrep 32

49 extraction and in 15 using the Nucleospin extraction. Upon re-extraction with an additional blood stain from the donor of the negative sample, ALAS2 was still not detected, but this was most likely due to some unknown sample issue (age, freeze-thawing, etc.) rather than the extraction method itself since no other problems were encountered with the blood samples. STATH was detected in all 10 of the Nucleospin saliva samples and initially in 8 of the AllPrep saliva samples. After re-amplifying the two samples with 5 µl of c instead of 3, a positive result was obtained for one of the samples. The other sample that was still negative was then amplified with HTN3 saliva primers to determine if it was a sample issue or a problem with the sensitivity of the marker used. HTN3 was then detected indicating a positive result. PRM2 was detected in 7 of the 8 semen samples for both the AllPrep and Nucleospin extractions, and the one sample that yielded a negative result was from a vasectomized male, so the protamine marker which is found in spermatozoa would not likely be present [4]. For the vaginal secretion samples, MUC4 was only detected in 3 of the 8 samples for both extraction methods. For samples with extract remaining, another SuperScript III RT reaction was performed with double the amount of RNA input because the quantitation result was likely elevated by bacterial RNA; however, no additional samples were positive for MUC4. A MUC4 singleplex was then performed to determine if the problem was caused by decreased primer sensitivity in the multiplex. MUC4 was detected in 3 additional samples for both extraction methods, making a total of 6 samples with positive results. An ABI High Capacity kit RT reaction was also performed, and VAG1 was detected in the remaining negative Nucleospin samples and in one additional AllPrep sample. For the one vaginal sample that was still negative, another AllPrep extraction was performed with new duplicate swabs, and MUC4 was detected in both samples. 33

50 AllPrep and Nucleospin extractions were also performed with ½ and ¼ vaginal swabs to see if reducing the sample size would allow for more efficient RNA recovery. Using a smaller sample size increased the number of positive results for the Nucleospin extraction, but none of the ½ or ¼ swabs from the AllPrep extraction were positive for MUC4 which may be caused by a combination of the low amount of RNA obtained using that extraction and the low sensitivity of the marker. For the menstrual blood samples, MMP-10 was detected in 2 of the 5 AllPrep samples and in 4 of the 5 Nucleospin samples. The samples from one donor for which a negative result was obtained with both extraction methods visually contained very little blood on the swab to start with, so it was not surprising that MUC4 was detected in that sample but MMP- 10 was not. The AllPrep extraction was repeated with new menstrual blood samples from the other two donors that yielded negative results, and MMP-10 was then detected in both samples indicating that the problem was sample dependent (day of menstrual cycle, amount of sample on swab, etc.). Sensitivity After the optimized protocols were tested using a larger number of samples, the sensitivity of the AllPrep and Nucleospin extractions was then compared. For blood, 10 µl, 5 µl, 1 µl, and 0.2 µl stains were extracted, and for saliva and semen, 50 µl, 25 µl, 10 µl, 5 µl, and 1 µl stains were extracted. Additional testing was done with ½ and ¼ buccal, semen, and menstrual blood swabs to be included with the ½ and ¼ blood stain and ½ and ¼ vaginal swab data already obtained, and full profiles were obtained for all of the ½ and ¼ swab samples (Table 5). Table 6 summarizes the and RNA detection results for the blood, saliva, 34

51 and semen stains from the AllPrep and Nucleospin extractions. Using a peak threshold of 100 RFUs, all of the saliva and semen stain samples, including the smallest amount tested (1 µl), produced full STR profiles. For the blood stain samples, full profiles were produced from all of the Nucleospin samples except for 0.2 µl stains from two donors which had 50% and 41% of the expected alleles present. For the AllPrep blood stain samples, full profiles were not produced from any of the 1 µl or 0.2 µl stains with 64% (2 samples) and 27% of alleles present for the 1 µl samples and 0%, 59%, and 32% of alleles present for the 0.2 µl samples. Figures 15, 16, and 17 show examples of the profiles obtained for the blood, saliva, and semen stains extracted using the AllPrep and Nucleospin extractions. As seen in Table 5, RNA was detected in all of the ½ and ¼ saliva and semen swab samples. MMP-10 was detected in three of the four ½ menstrual blood swab samples and two of the four ¼ swab samples from the AllPrep extraction and in all of the ½ and ¼ menstrual blood swab samples from the Nucleospin extraction. RNA was detected in all of the blood stain samples from the AllPrep extraction and in all of the samples from the Nucleospin extraction except for the 0.2 µl sample from one donor. Figure 12 shows an example of the RNA profiles obtained from the blood samples from one donor and both extraction methods. RNA was detected in all of the saliva stain samples from the Nucleospin extraction except for the 1 µl sample from one donor. For the saliva samples extracted with the AllPrep kit, only one 50 µl stain and one 25 µl stain sample were positive for STATH. All of the AllPrep saliva stain samples were amplified again using a HTN3 singleplex, and HTN3 was detected in an additional 50 µl and 25 µl stain sample. Figure 13 shows examples of the saliva RNA profiles obtained from one of the donors. For the semen stain samples, PRM2 was detected in all of the 35

52 Nucleospin and AllPrep RNA samples. Figure 14 shows examples of the RNA profiles obtained from one of the donors. 36

53 CHAPTER FOUR: RESULTS ( AND RNA STABILITY) Indoor Samples The NCFS co-extraction using Nucleospin columns was used to extract and RNA from blood, saliva, semen, and vaginal secretion stains (swabs) incubated at various temperatures in order to compare their relative degradation rates. Changes in and RNA yields over time did not follow a set trend and even increased for some of the later time points (Figures 18-21). The reason for this is unknown, but there may just have been a high level of inter and intra-donor variability. For the RNA samples, an increase in yield could indicate bacterial growth, although none was visibly present on the samples. yields from the saliva and vaginal secretion samples incubated at 56 C did tend to decrease for the later time points which was probably due to degradation of the (Figures 19 and 21). Room Temperature Dried blood (1/3 swab used), saliva (whole swab used), semen (whole swab used), and vaginal secretion (1/2 swab used) samples from three donors were extracted for the room temperature (RT) condition. Full profiles were obtained for all samples of each body fluid type out to 1 year and would likely have been obtained longer. For the blood samples, the RNA marker used (HBB) was also detected out to 1 year for all three donors. An ALAS2 singleplex was then used to determine if the same results would be obtained with a less sensitive blood marker. ALAS2 was only detected in a few of the samples and not even in all of the Time 0 control samples. MinElute columns were used to increase the peak heights and in some cases, 37

54 this allowed for ALAS2 to be detected in previously negative samples. It was then determined that the issues appeared to be arising from using a whole soaked blood swab which could be causing the column to become clogged or PCR inhibitors to co-purify with the RNA. The extractions were then repeated using 1/3 of a blood swab, and the results were greatly improved: ALAS2 was detected in all of the RT samples out to 1 year. An HBB singleplex and STR amplification was repeated with the 1/3 swab samples, and the same results were obtained. RNA markers were also detected in the saliva, semen (both markers), and vaginal secretions samples out to 1 year. No comparison of the stability of the and RNA in these stains could, therefore, be made because not enough degradation had occurred to affect the ability to obtain results (data not shown). 37 C The body fluid stains (swab) incubated at 37 C were then extracted. For the blood samples, full profiles were obtained and HBB was detected out to 1 year for all samples. ALAS2 was also detected out to 1 year but for only two of the three donors. Figure 22A shows the average and RNA profiling success rates for the blood samples incubated at this temperature. Table 7 also shows more detailed results (both RNA markers and all loci) for the three individual blood donors at the 1 year time point. Full profiles were obtained and both RNA markers were detected in the semen samples out to 1 year (data not shown). However, initially TGM4 was not detected in one of the 6 month samples. It was unclear why TGM4 was not detected since it was present in the other two 6 month samples and all three 1 year samples for this condition. An additional swab was extracted for this donor and time point, and both 38

55 PRM2 and TGM4 were then detected. Full profiles were obtained out to 6 months for the saliva samples with good partial profiles obtained for the three 1 year samples (average of 83% of expected alleles present). HTN3 was detected in the RNA samples out to 1 year for all three donors. Figure 23A shows the and RNA profiling success rates for the saliva samples, and Table 9 shows the detailed results for the three donors at the time point where some alleles were not detected. For the vaginal secretion samples, good partial profiles were obtained for two of the 1 year samples (28 or 29 of expected alleles) and one of the 6 month samples (29 alleles). MUC4 was detected out to 1 year (2 of 3 samples). Figure 25A shows the average lengths of time that and RNA could be successfully recovered and analyzed for the vaginal secretion samples, and Table 12 shows the detailed results for the three donors starting at the time point where alleles/muc4 were not detected. Again, for this condition it was difficult to compare and RNA stability because they were both still recoverable out to about 1 year. The partial profiles that were obtained were of good quality (contained the majority of expected alleles), and the RNA body fluid markers were detected in at least two of the three 1 year time point samples. 56 C and RNA from the body fluid stains (swabs) that had been incubated at 56 C were then extracted to determine if an increase in temperature would be more detrimental to and RNA stability. Full profiles were obtained out to 6 months for all of the blood samples except for one (31 alleles), and partial profiles (~51% of expected alleles) were obtained for the 1 year samples. HBB was still detected out to 1 year for all three donors while ALAS2 was detected out to 6 months (1 sample) or 3 months (2 samples). Figure 22B shows the and 39

56 RNA profiling success rates for the blood samples incubated at 56 C, and Table 8 shows the detailed results for the three donors at the time points where alleles/alas2 were not detected. The and RNA stability in these samples appeared to be similar because good partial profiles were obtained and HBB was detected at the 1 year time point, but detection of the less sensitive RNA marker was lost around 6 months. For the saliva samples, the results for the 56 C condition were slightly more variable among the three donors as can be seen in Table 10. Partial profiles were obtained for all of the 6 month (26% of alleles) and 1 year (21% of alleles) samples. A full profile was obtained for one of the 3 month samples while partial profiles were obtained for the other two donors (81% of expected alleles). HTN3 was detected in the saliva samples out to 1 year (2 donors) or 6 months (1 donor) for the 56 C condition. These results indicated that RNA was more stable than in the saliva stains since good profiles (majority of alleles present) were only obtained out to 3 months, but HTN3 could be detected out to 6 months or 1 year (Figure 23B). Partial profiles were obtained for two of the semen samples incubated for 1 year (18 and 26 of expected alleles), and no profile was obtained for the 6 month sample from one of those donors. This result did not fit with the rest of the data as full profiles were obtained for the other two 6 month samples. Another 6 month time point semen swab was extracted for this donor, and a full profile was obtained, so it is uncertain why the first sample had no profile, but it seems more likely that an error occurred during amplification or analysis of that particular sample. PRM2 and TGM4 were detected in the RNA samples out to 1 year (TGM4 detected in two of three 1 year samples). As seen in Figure 24, the recoverability of and RNA for the 40

57 semen samples did not differ greatly as both and RNA profiles were obtained out to 1 year. The detailed results for the three semen donors at the 1 year time point are shown in Table 11. For the vaginal secretion samples, partial profiles were obtained for all of the 6 month (47% of alleles) and 1 year samples (46% of alleles) for the 56 C condition. Additionally, a partial profile was obtained for the 1 month (14 alleles) and 3 month (8 alleles) samples incubated at 56 C from the third donor while full profiles were obtained at these time points for the other two donors. The 6 month and 1 year samples also appeared to be more degraded compared to the other two donors since less than 10 alleles were present. The MUC4 detection results seemed to correspond with the results because MUC4 was not detected in even the 3 week time point for the third donor but was detected out to 3 months for the other two donors (Table 13). In these samples, appeared to be more stable than RNA because a good profile was obtained at least one time point later than RNA was detected (Figure 25B), but this could in part be due to low sensitivity of the MUC4 marker. Substrates Stains made on other substrates were then tested to determine if there would be any effect on the stability of the and RNA compared to the samples collected on swabs. The substrates used were carpet and denim. Carpet was chosen because it is a synthetic material and also a likely substrate that a body fluid could be found on at a crime scene. Denim was chosen as another common substrate and also because the indigo dye used is known to be an inhibitor of PCR [51;52]. Since and RNA profiles could be obtained out to the 1 year time point (and 41

58 likely longer) for all of the swab samples incubated at room temperature, just the samples on denim and carpet for the 37 C and 56 C conditions were tested. Carpet In general, the results for the body fluid stains on carpet seemed to be similar to those obtained for the swab samples. For blood, full profiles were obtained, and HBB and ALAS2 were detected in all of the blood stains incubated at 37 C (Figure 26B, Table 14). For the 56 C condition, full profiles were obtained out to 6 months for two of the donors, but there seemed to be more degradation of the samples from the third donor since a full profile was only obtained out to 1 month (Table 15). ALAS2 was also not detected past 1 month for this donor but was detected out to 6 months and 1 year for the other two donors. HBB was detected in all samples out to 1 year. The average lengths of and RNA recoverability from the blood stains on carpet for the 56 C condition are shown in Figure 27B. It was not apparent if HBB stability was affected because the latest time point available to test was 1 year and HBB appeared to be stable beyond this point. For the saliva stains made on carpet, recoverability was slightly improved because full profiles were obtained out to 1 year for the 37 C condition (vs. 6 months), and better partial profiles were obtained at the 6 month time point (average of 26 vs. 8 alleles) for the 56 C condition. Figures 29 (37 C) and 30 (56 C) show the relative and RNA profiling success rates for the saliva stains made on the three substrates. The RNA results for the carpet samples were variable because there were some issues with HTN3 detection, but HTN3 was still detected out to 6 months or 1 year in at least one sample. For the 37 C condition, HTN3 was detected in 42

59 two of the three 1 year samples but was not detected in the Time 0 control sample for the third donor (Figure 29B). Another saliva marker, MUC7, was used to test some of the negative samples, and MUC7 was detected in the one negative 1 year sample but not the Time 0 sample (data not shown). For the 56 C condition, HTN3 was detected out to 1 year for one donor, but again, one of the Time 0 control samples was negative for both HTN3 and MUC7 (Figure 30B). This may be related to the sensitivity of the saliva markers used and potential loss of sample since the saliva stain was not visible on the tan carpet although a circle had been drawn around where the stains were made. A comparison to the saliva stains on swabs was difficult to make because of the inconsistent RNA detection results; however stability appeared to be improved. For the semen stains made on carpet, similar results were obtained for the carpet and swab samples for both the 37 C and 56 C conditions because and RNA still appeared to be relatively stable after 1 year. Full profiles were obtained and both PRM2 and TGM4 were detected out to 1 year for the 37 C condition (Figure 32B). Figure 33B shows the average and RNA profiling success rates for the semen stains on carpet for the 56 C condition. Partial profiles (30 and 27 alleles present) were obtained for two of the 1 year samples and a full profile was obtained for the third sample while PRM2 (all samples) and TGM4 (2 of 3 samples) were detected out to 1 year (Table 16). Denim For the blood stains made on denim, detection results similar to those for the swab and carpet samples were obtained for the and RNA (HBB) samples for the 37 C condition 43

60 (Table 14). However, recoverability for the 56 C condition was reduced with full profiles only obtained out to 1 month (1 donor) or 3 months (2 donors). Similarly, ALAS2 recoverability was decreased as it was not detected in any of the 1 year samples or in one of the 6 month samples for the 37 C condition. For the 56 C condition, ALAS2 was only detected out to 1 month (1 donor) or 3 months (1 donor). Figures 26 and 27 show the and RNA profiling success rates for the blood stains made on the three substrates for the 37 C and 56 C conditions, respectively, and Tables 14 and 15 show the detailed detection results for the three donors. Additionally, Figure 28 shows an example of the and RNA profiles (from one donor) obtained for the blood stains made on the three substrates incubated at 56 C for 1 year and any differences in recoverability observed for that donor: and RNA (HBB) were recoverable at 1 year for the swab sample, and RNA (HBB and ALAS2) were recoverable at 1 year for the carpet sample, and only RNA (HBB) was recoverable at 1 year for the denim sample. For the saliva stains made on denim, the relative RNA stability could not be determined because HTN3 was only detected in one Time 0 sample and 1 year sample for the 37 C condition and in one Time 0 sample for the 56 C condition. It appears that these poor results were due to PCR inhibition caused by the indigo dye present in denim [51]. The results were somewhat surprising since the ability to obtain a full profile did not seem to be negatively affected (Figures 29 and 30), and it seems reasonable to think that the Nucleospin and Nucleospin RNA columns would be similar in their ability to remove inhibitors and contaminants from the sample. There did not seem to be an issue with PCR inhibition in the blood RNA extracts, but the blood stain did not spread out as much on the denim, so a smaller piece of substrate could be used in the extraction. It is possible that since ALAS2 was not 44

61 detected in as many of the later time point samples, that it may have been slightly affected by inhibition, but there was no obvious effect on HBB amplification because it is a more sensitive marker. There are means of removing these PCR inhibitors, but for our purposes, we did not make any further attempts to do so, since it was not entirely unexpected that this issue could arise with a stain made on denim. Figures 29 and 30 show the and RNA recoverability from saliva stains on the three substrates for the 37 C and 56 C conditions, respectively. Figure 31 shows an example of the and RNA profiles obtained for the saliva stains made on the three substrates incubated at 56 C for 1 year. Detailed results for the donors are not shown because the RNA results were inconclusive, and an accurate comparison of and RNA stability could not be made. For the semen stains made on denim, full profiles were obtained out to 1 year for the 37 C condition, but for the 56 C condition, full profiles were only obtained out to 1 month (2 donors) or 6 months (1 donor) compared to 6 months or 1 year for the swab samples (Figures 32C and 33C). Again, there seemed to be some PCR inhibitors present in the RNA extracts since TGM4 was only detected in a couple of samples. PRM2 was detected out to 1 year for the 37 C condition but was not detected in one of the 6 month samples even after re-extraction. For the 56 C condition, PRM2 was detected out to 1 year for two donors but was not detected past 1 month for the third donor. Figures 32 and 33 show the and RNA profiling success rates for the semen stains on the three substrates for the 37 C and 56 C conditions, respectively, and Table 16 shows the detailed results for the three donors (56 C). Figure 34 shows an example of the and RNA profiles (from one donor) obtained for the semen stains made on the three substrates incubated at 56 C for 1 year and any differences in recoverability observed for that 45

62 donor: and RNA were recoverable at 1 year for the swab and carpet sample, and only RNA (PRM2) was recoverable at 1 year for the denim sample. Environmental Samples Outside Covered and Uncovered Blood, saliva, and semen stains and vaginal secretion swabs were placed outside, covered and uncovered, to determine how exposure to heat, sunlight, rain, and humidity would affect and RNA stability. For all of the body fluid stains, and RNA profiling success was better for the covered samples compared to the uncovered samples. This is not surprising since the exposure to rain appeared to wash away the majority of the stain. The yields tended to decrease as samples were exposed to the conditions for longer periods of time which could indicate either degradation or loss of sample; however, the RNA yields increased in many cases which was likely due to fungal or bacterial growth. This was especially apparent for the covered and uncovered blood samples (Figure 35). The detailed detection results for the four donors of the covered blood samples are shown in Table 14. Full profiles were obtained out to 3 months (all samples) and 6 months (1 sample). There was some variability in the RNA detection results with HBB being detected out to 4 weeks for two donors, 3 months for one donor, and 6 months for the last donor. ALAS2 was detected out to 1 week (3 donors) or 4 weeks (1 donor). For the uncovered condition, a full profile could not be obtained past 3 days and partial profiles (10 or 8 alleles) were only obtained out to 1 week. Again there was variability in the 46

63 RNA results, but in general HBB was detected longer than a profile was obtained (1 week, 4 weeks, and 6 months). ALAS2 was not detected past 1 day in the uncovered samples. Detailed detection results for the uncovered blood samples are shown in Table 15. The average HBB and ALAS2 peak heights are shown in Figure 36, and the average allele peak heights are shown in Figure 37. The peak heights for the RNA markers and alleles decreased as the length of exposure increased, and this occurred sooner in the uncovered samples (~3 days) compared to the covered samples (~3 months), indicating a loss of amplifiable product. Figure 38 shows that the relative and RNA recoverability was similar for the outside samples; however, HBB was detected longer than a good profile was obtained for the uncovered samples (Figure 38B). The and RNA profiles obtained from one of the donors for the Time 0 control and also the time points where alleles, the body fluid markers, or both were not detected are shown in Figures 39 (covered) and 40 (uncovered). For this donor, RNA (HBB) was recoverable for a longer period of time than. The and RNA recovery from the outside saliva stains followed a similar trend as the blood samples with yields decreasing with each time point. The RNA yields remained somewhat constant or decreased but increased again by 4 weeks indicating the RNA originated from the environment (Figure 41). The detailed detection results for the four donors of the covered saliva stains are shown in Table 16. A full profile was not obtained past 1 week, and there was variability among the donors with partial profiles starting at 1 day (31 alleles) for one donor and at 1 week (29 alleles) for the second and third donors while full profiles were obtained out to 1 week for the fourth donor. There was also variability in the RNA results with HTN3 being detected only in the control sample (1 donor), out to 1 day (2 donors), or out to 1 47

64 week (1 donor). For the uncovered saliva stains, full profiles were obtained at the 1 day time point for all donors and partial profiles were obtained out to 1 week (11 alleles present). HTN3 was not detected beyond the Time 0 control for two donors and was detected out to 1 day and 3 days for the other two donors. Due to these somewhat poor RNA detection results, further attempts were made to determine if HTN3 could be detected in any additional samples. The RNA quantitation results for these samples would likely have been elevated due to the presence of bacterial RNA, so the samples were amplified again using 5µL and 8µL of c in the singleplex reaction. For one of the donors, HTN3 was detected in the 3 day sample using 5µL of c and in the 4 week sample using 8µL of c. The samples were also purified using MinElute columns to see if the results could be improved any further, and peak heights were increased, however, no additional samples were positive for HTN3. Detailed detection results of the uncovered saliva stains are shown in Table 17. The average HTN3 peak heights are shown in Figure 42, and the average allele peak heights are shown in Figure 43, and again, peak heights decreased with time (starting around 3 days) with a noticeable decrease in HTN3 peak heights occurring sooner for the uncovered stains (1 day). The and RNA profiling success rates for the outside saliva stains are shown in Figure 44. RNA (HTN3) stability in the outside stains seemed to be affected more by the elements compared to the in these stains, especially considering that RNA appeared to be more stable than in the saliva samples incubated at the set indoor temperatures. and RNA profiles from one of the donors are shown in Figures 45 (covered) and 46 (uncovered) for the Time 0 control and also the time points where alleles, HTN3, or both were not detected: appeared to be more stable in the covered 48

65 sample, but both and RNA were detectable to about the same time point in the uncovered sample. The change in and RNA yields from the semen stains were similar to the saliva stains with increased exposure accompanied by a decrease in the amount of obtained while the RNA yields stayed about the same or increased (Figure 47). As seen in Table 18, full profiles were not obtained past 1 week for the covered semen stains. Weak partial profiles (5 and 3 alleles) were obtained for the 4 week samples. PRM2 was detected out to 1 week for both donors with a small 50 RFU peak for one of the 4 week samples. In addition to the PRM2/TGM4 duplex, the samples were also amplified with a TGM4 singleplex using a different reverse primer that would result in a larger amplified c product. These primers were used because if happened to be present in the samples, the normally used TGM4 primers could possibly amplify it, and the product would be of the same size as if a true mrna transcript was present. Using these two different primer sets, TGM4 was detected out to 1 day and 1 week for the two donors. For the uncovered semen stains, a full profile was not obtained past 1 day with partial profiles obtained out to 3 days (19 alleles) or 1 week (3 alleles). PRM2 was detected out to 3 days, and TGM4 was detected out to 1 day or 3 days. Detailed detection results for the uncovered semen stains are shown in Table 19. The average PRM2 and TGM4 peak heights for the covered and uncovered conditions are shown in Figure 48, and the average allele peak heights are shown in Figure 49. A decrease in PRM2 peak heights occurred later (~4 weeks vs. 1 week) or at about the same time (~3 days) as a decrease in allele peak heights for the covered and uncovered conditions. The relative and RNA profiling success rates for the outside semen stains are shown in Figure 50, and the results indicate that and RNA stability was very similar in the 49

66 semen stains. and RNA profiles from one of the donors are shown in Figures 51 (covered) and 52 (uncovered) for the Time 0 control and also the time points where alleles, the body fluid markers, or both were not detected. For the donor shown, RNA was recoverable for one time point later than. Again, the yields from the vaginal secretion swabs decreased with increased exposure and the RNA yields increased for one donor while they decreased for the other donor (Figure 53). For the covered vaginal secretion samples, full profiles were obtained out to 1 day with partial profiles for the 3 day (23 alleles), 1 week (17 alleles), and 4 week samples (8 alleles). MUC4 was only detected in the 1 day sample from one donor. Results were somewhat similar for the uncovered samples, with MUC4 being detected in the 1 day sample from the same donor. Partial profiles were obtained for the uncovered samples for all time points except for one of the 4 week samples (no profile) with the following allele percentages: 63% (1 day), 27% (3 days), 25% (1 week), and 3% (4 weeks). The RNA samples were then amplified a second time for the same reason that the saliva samples were (bacterial RNA) using 5µL of c, and MinElute purification was also performed, but MUC4 was not detected in any additional samples. Another RT reaction was then performed using double the amount of sample input, and MUC4 was detected in the covered 1 day sample for the second donor and in the uncovered 3 day sample for the first donor. Detailed detection results for the two donors are shown in Tables 20 and 21. The average MUC4 peak heights are shown in Figure 54, and the average allele peak heights are shown in Figure 55, and peak heights had already decreased after 1 day of exposure. The average profiling success rates are shown in Figure 56, and similar to the indoor vaginal secretion samples, seemed to be more stable than the RNA in these swabs. and RNA 50

67 profiles from one of the donors are shown in Figures 57 (covered) and 58 (uncovered) for the Time 0 control and also the time points where alleles, MUC4, or both were not detected. For the covered sample, appeared to be more stable; however, for the uncovered sample, RNA was detected longer than a good profile was obtained. Other Conditions Additional blood, saliva, and semen stains (1 donor per fluid) on cotton were incubated in the shade, sun, and on a patio for varying amounts of time. The amount of and RNA recovered from these samples can be seen in Figures 59 (blood), 60 (saliva), and 61 (semen). Similar to the other outside conditions, the amount of RNA recovered from the stains left in the shade and sun tended to stay the same or increased while yields decreased with time. However, for the stains left on a patio, both and RNA yields either increased or remained approximately the same which is not surprising since these stains would have been exposed to fewer environmental conditions. The detection results for the three body fluids were similar in that and RNA profiles were generally obtained the longest for the stains left on a patio. For the blood stains, and RNA recoverability was somewhat similar, and the and RNA profiling success rates can be seen in Figure 62. Full STR profiles were obtained for the samples left in the shade and sun for 1 day and 2 days, and a partial profile was obtained for the 1 week time points (8 alleles). HBB was detected out to 1 week for both the sun and shade samples but not detected by 1 month. ALAS2 was detected out to 2 days for the shade samples but was not detected in any of the samples left in the sun. Full profiles were obtained out to 1 month for the patio samples, and after a few additional time points were extracted for this 51

68 condition (2 months, 3 months, and 4 months), a full profile could be obtained out to 3 months with a good partial profile obtained for the 4 month sample (31 alleles). HBB and ALAS2 were also detected out to 4 months for the patio samples (3 month sample was negative for ALAS2). The and RNA profiling success rates for the saliva samples are shown in Figure 63. Full profiles were obtained out to 2 days for the shade samples with a partial profile containing only 2 alleles was obtained for the 1 month time point. RNA appeared to be more stable in these samples as HTN3 was detected out to 1 month. However, similar to ALAS2 in the blood samples, HTN3 was not detected in any of the sun samples while good profiles were obtained out to 2 days (19 alleles). and RNA stability was similar in the patio samples because full profiles were obtained and HTN3 was detected out to 1 week. Figure 64 shows the and RNA profiling success rates for the semen stains. For all three conditions, and RNA were recoverable for about the same amount of time. Good profiles were obtained out to 12 days for the shade samples, and PRM2 and TGM4 were detected out to 12 days and 2 days, respectively. Good profiles were obtained out to 2 days for the sun samples, and PRM2 and TGM4 were both detected out to 2 days. For the patio samples, full profiles were obtained out to 12 days, a good partial profile was obtained for the 1 month sample (22 alleles), and PRM2 and TGM4 were detected out 1 month. 52

69 CHAPTER FIVE: DISCUSSION /RNA Co-extraction analysis has been used in forensic science for several years now to obtain a genetic profile from biological material left at a crime scene for comparison to reference samples/databases [1]. Several serological based tests are routinely used to identify the type of body fluid or fluids present, but these tests are not always sufficiently sensitive or specific, confirmatory tests do not exist for all body fluids, and they must be performed in a sequential manner. Being able to utilize mrna body fluid typing in conjunction with analysis has great promise in forensic biology because of the similarity of the molecules and therefore, the procedures used to analyze them. Co-extracting the two nucleic acids from the same biological stain allows for less time and consumption of sample than would be required to perform two separate extractions. Of the co-extraction methods that were evaluated, the in-house NCFS co-extraction method tended to produce the highest yield for both and RNA, and and RNA profiles were obtained (except for one blood sample which likely contained hematin). The two coextractions, ToTALLY RNA and TRIzol, which utilize RNA extraction with the back extraction of from the organic phase, produced adequate RNA yields, but there were issues with detecting RNA markers in the blood samples. Since these are organic extractions and no spin column purification was used, it is likely that hematin remained in these samples, as with the NCFS co-extraction, causing the negative RNA results [40]. Relatively low yields were also obtained compared to the other methods, but there was still enough to produce full STR 53

70 profiles. Fleming and Harbison, in an attempt to co-extract and RNA, tested a method which used the back extraction of from the organic phase, but upon further investigation, they found that there was actually more in the aqueous phase with the RNA [6], and the same could have been true for the ToTALLY RNA and TRIzol extractions. The two methods that utilize spin columns, the AllPrep /RNA Mini kit and Chaos buffer/spin column extraction, produced relatively better yields but low RNA yields, and positive RNA results were not obtained for both blood samples. This is most likely due to the fact that these protocols are not optimized for typically encountered forensic samples. Based on these results, the NCFS co-extraction and AllPrep /RNA Mini kit were then optimized, the AllPrep kit being chosen because it is a spin column-based co-extraction method that can be automated and would be faster and more efficient than an organic method. The RNeasy Micro and AllPrep extraction protocols were designed to extract RNA or RNA and from tissues and cell samples which are homogenized prior to being transferred to the column. A forensic sample collected on a swab or other substrate cannot be homogenized in the same way and doing so could damage the or RNA (which may already be partially degraded). However, simply adding the lysis buffer provided with the kit to the sample and then applying it to the column did not seem sufficient to thoroughly release the and RNA into solution. For this reason, the various incubation times and temperatures were used to determine if incubating the sample in the buffer for longer periods of time or at a higher temperature, as with other extraction methods, would improve lysis and increase the yields obtained using a spin column extraction method. For the RNeasy Micro extraction, the amount of RNA recovered was increased when the samples were incubated in the lysis buffer for 1 hour at 56 C prior to 54

71 extraction. The same was observed for the AllPrep /RNA Mini extraction with 3 hours at 56 C allowing for the greatest increase in product. A concern with the AllPrep kit was the low RNA yields from the vaginal secretion samples especially since the yields were higher when using the RNeasy Micro kit. Due to this difference, it was thought that the RNA was likely binding to the column. The fraction was tested for RNA and VAG1 was detected in some samples, and the Ribogreen quantitation revealed high amounts of RNA in the fraction from vaginal samples. In the AllPrep manual, it states that RNA may bind to the columns if the sample is sufficiently acidic [53]. It is possible that the vaginal secretion samples have a lower ph which is why more RNA was found in the fraction than with the other body fluids that have a more neutral ph. Since the number of positive results obtained with the AllPrep extraction was similar to that obtained with the NCFS co-extraction using Nucleospin columns, further attempts to rectify this problem were not taken. If more RNA was required, a similar procedure could be used by taking a fraction of the eluate, treating it with DNase, and then combining it with the RNA sample, or the column could be washed with Buffer RLT after the RNA-containing flow through is collected to elute more RNA that may still be bound to the column membrane [23]. The Nucleospin Genomic Clean-up XS kit is designed to provide a fast purification method while recovering high amounts of concentrated from small sample sizes which makes it ideal for use with forensic samples, and the same is true for the Nucleospin RNA Clean-up XS kit [54;55]. Hudlow, et al. showed that first diluting the sample with TE -4 buffer and then adding the NT binding buffer at a ratio of 4:1, resulted in the best yields which is the ratio that was used here [49]. Compared to the standard NCFS co-extraction, 55

72 yields from some of the body fluids were reduced when using the Nucleospin columns (especially from blood), but the and RNA appeared to be of high quality and free of PCR inhibitors. Even with some of the lower yields, the sensitivity of the extraction method and the downstream reactions was still sufficient to produce high and RNA profiling success rates. Some of the other alterations to the protocol were tested to determine if lysis could be improved or if more /RNA could be segregated into the aqueous phase by changing the ph of the phenol/chloroform reagent [15], but we did not find any of these alterations to be a significant improvement to the protocol. Some issues with the RNA detection in vaginal and menstrual blood samples were encountered even with those samples extracted using standard RNA extraction procedures. For the menstrual blood samples, it may be that consistent detection results were not obtained because the amount of sample on the swab was donor dependent, and often times, markers for blood or vaginal secretions were detected while the menstrual blood marker was not [56]. With both of these sample types, there is endogenous bacterial RNA in the vagina that is also extracted [57], and there is not currently a human-specific RNA quantitation method, so it is difficult to know the true amount of target RNA being used in downstream reactions. Additionally, development of sensitive and specific vaginal secretion RNA markers is still in progress, so the sensitivity of the vaginal marker used in the multiplex may not have been sufficient to always produce positive results, as was observed when MUC4 was detected in more samples when a singleplex was used. MUC4 was mostly used in this study, but it can sometimes be detected in saliva as well which does not make it an ideal vaginal secretion marker [11;57]. Despite these 56

73 limitations, after optimization of the co-extraction protocols, the detection results for the vaginal and menstrual blood samples were improved. After the optimization of the two chosen co-extraction methods, both appeared to be acceptable for forensic use with high and RNA profiling success rates. The AllPrep /RNA Mini kit provides an automatable method with less sample manipulation, but the NCFS co-extraction with Nucleospin columns uses both organic and spin column purification which would be suitable for stains on difficult substrates potentially containing more contaminants. The sensitivity of both methods was also surprisingly good considering the fact that they are co-extraction methods, but the ability to obtain profiles also depends on the downstream reactions and the sensitivity of the RNA markers used which was limiting in some cases. Positive and RNA detection results could be obtained for at least one donor using the lowest amount of blood (0.2 µl) and saliva and semen (1 µl) tested. The Nucleospin extraction appeared to be more sensitive than the AllPrep extraction (Tables 5 and 6). Issues were encountered with the saliva stain samples that were extracted using the AllPrep kit as HTN3 or STATH was only detected in the 50 µl and 25 µl stain samples. It is uncertain why these poor results were obtained since the saliva stains used were made at the same time and from the same liquid sample for both the AllPrep and Nucleospin extractions. Both extraction methods performed well with the ½ and ¼ swab and stain samples with more positive RNA detection results obtained for the menstrual blood swabs when using the Nucleospin extraction. 57

74 and RNA Stability After the co-extraction methods were optimized, one could then be used to compare the relative stability of and RNA in the same stain. We wanted to determine if and RNA degrade at relatively the same rate in a dried body fluid stain or if one degraded more quickly than the other. It has often been thought that RNA is less stable than, and while this may be true in vivo, it is not always the case. Recent studies have shown mrna markers for body fluid identification are sufficiently stable for use in forensic science [29;30;32;33;58]. However, there has not been a direct comparison of the degradation rates of and RNA across various conditions and body fluids. Dried blood, saliva, semen, and vaginal secretion samples were incubated at various set temperatures as well as outside to determine at what point and RNA detection was no longer possible and if one could be detected longer than the other. It is somewhat difficult to make a direct comparison of and RNA stability using only CE analysis since the presence of the mrna of interest is confirmed solely by one peak while a full profile requires allele peaks to be present for all loci examined. Even though a full profile is desirable for making comparisons, a good partial profile can still provide adequate information for making suspect exclusions [1], but if no RNA peak is observed, the body fluid(s) present could remain inconclusive. For this reason a profile with the majority of the expected alleles present was considered to be a good profile in the /RNA stability comparison. A detection threshold of 50 was employed meaning that no allele/rna marker peaks below 50 RFUs would be designated. However, in some partial profiles, peaks were visible that were below this threshold (Tables 19, 22, 25, and 28). Although 50 RFUs is common, labs may use different thresholds, and while using caution, some analysts would call 58

75 these small peaks as alleles, thereby providing better partial profiles than what is reported here. It should, therefore, be understood that there may have been additional genetic information in the partial profiles, but we wanted to keep the results consistent by not calling alleles or RNA marker peaks below 50 RFUs even if they were visible. Often times, especially with the outside samples, there was not sufficient available to input the required amount into the PCR reaction, and when low levels of potentially degraded are used, stochastic effects such as allele drop-out, allele drop-in, peak imbalance, and increased stutter peaks as well as background noise from the instrument can make it difficult to interpret results [59;60]. We were working with known profiles, but in a forensic setting where the profile of the donor is not known, these types of factors would become much more problematic when interpreting results. This is why a 50 RFU threshold was used. The conditions under which the body fluid stains were found determined how long and RNA could be recovered and successfully analyzed. Stains were first incubated at set temperatures to see how long and RNA would remain stable when exposed to heat. Data has shown that lower temperatures are favorable to and RNA stability, so it was not unexpected that increasing the temperature to 56 C resulted in decreased stability [25;34;61;62]. We used this temperature, which is not a commonly encountered terrestrial temperature, as a way to artificially degrade the and RNA since achieving a significant amount of degradation at 37 C would have taken a greater length of time. For these indoor conditions, a difference in and RNA recoverability was not immediately apparent, especially for the RT and 37 C conditions as little to no reduction in recoverability was observed. For the 37 C condition, the only noticeable difference in recoverability was for one vaginal secretion sample for which a 59

76 good profile was obtained at 1 year but MUC4 was not detected. The degradation process was still relatively slow at 56 C as and RNA profiles were obtained after 6 months or 1 year. Both and RNA were still stable at 1 year in the blood and semen stains (swab). For saliva, good profiles were only obtained out to 3 months, but RNA was detected out to 6 months or 1 year. The opposite trend was observed for the vaginal samples, with profiles obtained out to 1 year, 6 months, and 3 weeks for the three donors while MUC4 was detected out to 3 months for the first two donors but was not detected past Time 0 for the third donor. As previously stated, this apparent instability of MUC4 may be related to the sensitivity of the primers or the relative abundance of the marker within the body fluid (which may also be donor/sample dependent). For the environmental samples exposed to more variables, a decline in and RNA stability occurred for all body fluids tested. The presence of microbiota, exposure to UV light, increased moisture, and higher temperatures all have been shown to negatively affect and RNA stability [29;33-35;61-63]. The dried state of a stain does reduce the occurrence of hydrolysis, and some enzymes that would normally break down and RNA become denatured during the drying process, but increased humidity promotes hydrolytic cleavage and can encourage bacterial growth which introduces exogenous nucleases [1;37;62;63;65]. With the outside uncovered samples, the greatest challenge was the exposure to rain which may not necessarily have resulted in degradation of the sample but simply sample loss in which case, partial profiles may have been a result of low copy number rather than degradation. That being said, alleles for the larger loci were generally lost first which is indicative of degradation since amplification of shorter sequences of is more successful in degraded samples [63]. 60

77 For the blood stains, the results were variable among the donors: was recoverable out to about 3 months while RNA was detected out to 4 weeks, 3 months (2 donor), or 6 months (1 donor) for the outside covered condition. profiles were obtained out to 1 day or 3 days, and RNA was detected out to 3 days, 1 week, 4 weeks, or 6 months for the uncovered blood samples. Analysis of the blood stains left in the shade, sun, and on a patio showed similar results with HBB being detected as long as or longer than a good profile was obtained. Others have also observed that HBB can be detected longer than a profile can be obtained in samples exposed to the environment [8]. and RNA stability was relatively similar in the semen stains with both and RNA being recovered on average out to 1 week (covered), 3 days (uncovered), 12 days (shade), 2 days (sun), and 1 month (patio). For the saliva stains, the harsher conditions (outdoors covered/uncovered, sun) seemed to have more of an effect on the ability to detect HTN3, but RNA was more stable than (indoors, shade) or as stable as (patio) when exposure to environmental conditions was reduced. For the covered vaginal samples, good profiles were obtained out to 3 days or 1 week, but RNA was only detected at the 1 day time point; however, for the uncovered samples, good profiles were only obtained at 1 day while RNA was detected out to 3 days for one donor. The substrate on which the stains were made had a small effect on the relative level of and RNA degradation. A previous study comparing the effects of substrate on recovery and quality was performed with blood stains, and they found that substrates that the blood could soak into resulted in less recovery, and if the substrate was chemically treated, the level of impurities in the was also higher [52]. Synthetic carpet fibers are glued to an under layer which could impart chemicals into the sample, and the carpet could also be treated 61

78 with fungicides and bactericides [52]. The spin columns used with the optimized NCFS coextraction method should have removed these impurities potentially found in the carpet and also the indigo dye found in denim. Overall, the results for the stains on swabs and carpet were similar, but there were issues with apparent PCR inhibition in the stains made on denim. For blood, ALAS2 stability seemed to be improved for the carpet samples (6 months 1 year) while stability was about the same (good profiles at 1 year), and both ALAS2 and stability were decreased for the denim samples (to 3 months and 6 months) while no change in HBB stability was observed. For the saliva samples, the results were somewhat inconclusive due to PCR inhibition in the denim samples, but this inhibition seemed to be limited to RNA (c) amplification rather than STR amplification. stability was improved for the carpet samples (3 months 6 months) while HTN3 was still detected out to 1 year. There was also some variability in RNA detection results for the saliva stains on carpet, and this may be due to sample loss caused by the saliva soaking into the substrate, and the stain was not visible on the tancolored carpet like the blood and semen stains were. For the semen samples, the relative and RNA stability remained approximately the same for the carpet and denim samples compared to the swab samples (1 year); however, there appeared to be some PCR inhibition of PRM2 and TGM4 amplification in the denim samples. Based on these results, the substrate seemed to have a small effect on relative and RNA stability. Generally, if stability was decreased (denim) or increased (carpet), both and RNA were affected or a change in RNA stability could not be determined because of PCR inhibition or because the mrna marker was stable beyond the latest time point that was available for testing. It would be useful to examine stains made on 62

79 additional non-absorptive substrates and also incubate the stains for longer than 1 year for a better understanding of what effects, if any, the substrate has on relative and RNA stability. In summary, the degradation rates of and RNA seemed to coincide for the most part but varied slightly depending on the body fluid, conditions, and RNA marker used. In general, and RNA were successfully analyzed longer for the blood and semen samples compared to the saliva and vaginal secretion samples. This difference in recoverability among the body fluids may be due to the level of protection that the nucleic acids are given in terms of cellular components and also the relative amount of microbial activity. HTN3 is thought to be a secreted mrna in saliva which could possibly mean reduced protection from degradation, but the HTN3 marker has been shown to be sufficiently stable in dried saliva stains, which is consistent with the results for the RT, 37 C, and 56 C conditions [9;30]. It may be that naturally occurring microbiota in saliva and vaginal secretions played a role in the increased degradation of the and RNA in these stains which would have been augmented for the samples left outside. As previously observed, the environmental conditions that the stain was exposed to seem to have more effect on the degradation of and RNA as opposed to the age of the stain [32;61]. Compared to the indoor samples, recoverability was significantly reduced for all of the body fluid samples when increased humidity, UV light, and rain were introduced. The ability to detect RNA also depended on the RNA marker used, and it has been proposed that different mrna species degrade at different rates [27;30]. The differences in observed RNA marker sensitivity could be due to a number of factors, including relative abundance within the body fluid, differences in PCR efficiency, differences in amplicon length, and actual differences in the degradation rates of the species. 63

80 CHAPTER SIX: CONCLUSION The goals of this research were to evaluate available methods for the co-extraction of and RNA from the same biological sample, determine which method appeared to be the most suitable for forensic samples, optimize the chosen method to improve yields and achieve consistent detection results, and ultimately use the optimized method to determine the relative stability of and RNA in the same stain. Several co-extraction methods exist but have either not been thoroughly validated or are not designed for samples of low quantity or quality often encountered in forensics. By comparing several of these methods, we showed that and RNA can be successfully isolated from the same biological stain. Two of these methods, our inhouse NCFS co-extraction method as well as a commercially available kit, were then optimized and improved. High quality RNA was obtained with both of the optimized methods, and the ability to obtain full STR profiles was not negatively affected by co-extracting RNA, as high quality profiles were also produced. Even when extracting low amounts of degraded and RNA, the sensitivity of the Nucleospin extraction proved to be sufficient. Forensic laboratories may have different preferences when it comes to the extraction method employed, so by optimizing two different extraction types, we provide two suitable co-extraction methods which can be chosen based on the sample type or the needs of the lab. By using one of the optimized co-extraction methods, we were able to gain insight into the relative stability of and RNA in dried body fluid stains. In general, and RNA stability was found to be similar with a loss in ability to obtain a or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no profile was obtained, indicating that RNA in dried stains is 64

81 sufficiently stable for mrna body fluid typing to be used in forensic casework. The dried state of the stain allows for this increased stability of RNA, and during the extraction process, the RNA remains protected from degradation because RNases are removed, inactivated, or destroyed. The times when RNA appeared to be less stable than may have depended on the sensitivity of the RNA marker used and did not necessarily mean that the RNA was truly degraded beyond the point of recoverability. Further investigation would be necessary to fully understand the degradation process that is occurring in these stains on a molecular level (i.e. when degradation of the molecules begins to occur, what type of degradation is occurring, etc.), but there does not seem to be any evidence that suggests that mrna profiling should not be utilized in forensic casework based solely on the supposed instability of the molecule. 65

82 APPENDIX A: FIGURES 66

83 A Total Yield (ng) RNA AllPrep ToTALLY RNA TRIzol Chaos buffer NCFS Coextraction Extraction Method Standard Organic Extraction Standard Spin Column Extraction B Total Yield (ng) RNA AllPrep ToTALLY RNA TRIzol Chaos buffer NCFS Coextraction Standard Organic Extraction Standard Spin Column Extraction Extraction Method Averages are based on the amount of and RNA recovered from two 50 µl blood stains (A) and two buccal swabs (B). Each extraction method was performed twice with samples from two different donors (blood) or the same donor (saliva). Real time PCR (Quantifiler Human) and Ribogreen (RNA) were used for quantitation. Figure 1: Evaluation of and RNA Recovery Using the Five Co-extraction Methods and Standard and RNA Extraction Methods 67

84 Electropherograms show example of the RNA results for the blood (left) and saliva (right) samples. A 5-plex amplification reaction was used for body fluid typing. The RNA marker detected is indicated. (A) AllPrep (B) TRIzol (C) ToTALLY RNA (D) Chaos Buffer (E) NCFS Co-extraction. X-axis represents the size of the amplified fragment in base pairs and the y-axis represents relative fluorescent units. Figure 2: RNA Detection Results for Blood and Saliva Samples Extracted Using the Five Coextraction Methods 68

85 Full profiles obtained (SGM Plus ) from blood stains extracted using the co-extraction methods. Allele designations at each locus are indicated. X-axis represents the size of the fragment in base pairs and the y-axis represents relative fluorescent units. (A) AllPrep (B) TRIzol (C) ToTALLY RNA (D) Chaos Buffer (E) NCFS Coextraction. Figure 3: Profiles Obtained from Blood Samples Using the Five Co-extraction Methods 69

86 Full profiles (SGM Plus ) obtained for saliva samples extracted using the co-extraction methods. Allele designations at each locus are indicated. (A) AllPrep (B) TRIzol (C) ToTALLY RNA (D) Chaos Buffer (E) NCFS Co-extraction. Figure 4: Profiles Obtained from Saliva Samples Using the Five Co-extraction Methods 70

87 A Total Yield (ng) B Total Yield (ng) Standard 1 hour at RT 3 hours at Overnight 1 hour at RT at RT 37ºC Conditions 3 hours at Overnight 1 hour at 37ºC at 37ºC 56ºC 3 hours at Overnight 56ºC at 56ºC 0 Standard 1 hour at RT 3 hours at Overnight 1 hour at RT at RT 37ºC Conditions 3 hours at Overnight 1 hour at 37ºC at 37ºC 56ºC 3 hours at Overnight 56ºC at 56ºC RNA was extracted from duplicate blood stains and buccal swabs using the RNeasy Micro kit. After adding Buffer RLT, samples were either immediately transferred to the spin column (standard) or incubated in the lysis buffer for 1 hour, 3 hours, and overnight at room temperature, 37 C, and 56 C. Graphs represent average total RNA yields for the blood (A) and saliva (B) samples. Figure 5: Effect of Lysis Incubation Time and Temperature on RNA Recovery 71

88 A Total Yield (ng) Std Conditions 3 hours at 56oC B Blood Saliva Semen Vaginal Secretion Menstrual Blood Body Fluid Total Yield (ng) Std Conditions 3 hours at 56oC Blood Saliva Semen Vaginal Secretion Menstrual Blood Body Fluid The AllPrep /RNA Mini kit was used to extract and RNA from duplicate blood stains, saliva, semen, vaginal secretion, and menstrual blood swabs. The extraction was performed following the standard protocol or with a lysis incubation of 3 hours at 56 C. Graphs show the average total (A) and RNA (B) yields. Figure 6: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the AllPrep /RNA Mini Kit 72

89 A Total Yield (ng) Std Conditions Nucleospin columns B Blood Saliva Semen Vaginal Secretion Menstrual Blood Body Fluid Total Yield (ng) Std conditions Nucleospin columns 0 Blood Saliva Semen Vaginal Secretion Menstrual Blood Body Fluid The NCFS co-extraction was used to extract and RNA from duplicate blood stains, saliva, semen, vaginal secretion, and menstrual blood swabs. The extraction was performed using standard conditions or with Nucleospin and RNA spin columns. Graphs show the average total (A) and RNA (B) yields. Figure 7: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the NCFS Co-extraction 73

90 A B c was amplified using an mrna body fluid typing 6-plex reaction. RNA markers for blood, saliva, semen, vaginal secretions, and menstrual blood and amplified c fragment sizes are indicated. Samples were extracted with the NCFS co-extraction (A) with Nucleospin columns and the AllPrep Mini extraction (B) using the 3 hours at 56ºC lysis incubation. Figure 8: RNA Detection Results Using the Optimized Protocols of the NCFS Co-extraction and AllPrep /RNA Mini Extraction 74

91 A B 75

92 Complete profiles obtained from blood, saliva, semen, vaginal secretions, and menstrual blood samples using the NCFS co-extraction (A) with Nucleospin columns and the AllPrep extraction (B) using the 3 hours at 56ºC lysis incubation. Figure 9: Profiles Obtained Using the Optimized NCFS Co-extraction and AllPrep /RNA Mini Protocols 76

93 A Total Yield (ng) Nucleospin AllPrep Blood Saliva Semen Menstrual Blood Vaginal Secretion Body Fluid B Total Yield (ng) Nucleospin AllPrep Blood Saliva Semen Menstrual Blood Vaginal Secretion Body Fluid Blood (n=16), saliva (n=10), semen (n=8), vaginal secretions (n=8), and menstrual blood (n=5) samples were extracted using the optimized NCFS co-extraction and AllPrep /RNA protocols. Graphs show average total (A) and RNA (B) yields. Figure 10: Comparison of and RNA Recovery Using the Optimized NCFS Co-extraction and AllPrep Extractions 77

94 % Positive Results Nucleospin AllPrep 0 BL SA SE MB VS BL SA SE MB VS Body Fluid Percent of samples for which full STR profiles were obtained (; SGM Plus ) and percent of samples with corresponding body fluid marker detected (RNA; 6-plex). Profiling results obtained from testing of the optimized NCFS co-extraction and AllPrep /RNA Mini protocols with blood (BL, n=16), saliva (SA, n=10), semen (SE, n=8), vaginal secretion (VS, n=8), and menstrual blood (MB, n=5) samples. Figure 11: Profiling Success Rate of Optimized Protocols 78

95 A B Sensitivity of the optimized co-extraction methods. Blood stains decreasing in size were extracted (10 µl, 5 µl, 1 µl, and 0.2 µl). Samples were amplified using an mrna body fluid typing 6-plex reaction. The RNA marker detected and size of the amplified c fragment are indicated. (A) AllPrep (B) NCFS co-extraction. Figure 12: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood (RNA) 79

96 A B Sensitivity of the optimized co-extraction methods. Saliva stains decreasing in size were extracted (50 µl, 25 µl, 10 µl, 5 µl, and 1 µl). Samples were amplified using an mrna body fluid typing 6-plex reaction. The RNA marker detected and size of the amplified c fragment are indicated. (A) AllPrep (B) NCFS co-extraction. Figure 13: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva (RNA) A B Sensitivity of the optimized co-extraction methods. Semen stains decreasing in size were extracted (50 µl, 25 µl, 10 µl, 5 µl, and 1 µl). Samples were amplified using an mrna body fluid typing 6-plex reaction. The RNA marker detected and size of the amplified c fragment are indicated. (A) AllPrep (B) NCFS co-extraction. Figure 14: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen (RNA) 80

97 A B Blood stains decreasing in size were extracted (10 µl, 5 µl, 1 µl, and 0.2 µl). Examples of the profiles obtained are shown. (A) AllPrep (B) NCFS co-extraction. Figure 15: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood () 81

98 A B Saliva stains decreasing in size were extracted (50 µl, 25 µl, 10 µl, 5 µl, and 1 µl). Examples of profiles obtained are shown. (A) AllPrep (B) NCFS co-extraction. Figure 16: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva () 82

99 A B Semen stains decreasing in size were extracted (50 µl, 25 µl, 10 µl, 5 µl, and 1 µl). Examples of the profiles obtained are shown. (A) AllPrep (B) NCFS co-extraction. Figure 17: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen () 83

100 Total Yield (ng) RNA RT 37 C 56 C Time The NCFS co-extraction with Nucleospin columns was used to extract and RNA from blood stains (swab) from 3 donors incubated for various amounts of time at RT, 37 C, and 56 C. Real time PCR (Quantifiler Human) and Ribogreen RNA quantitation were used for and RNA quantitation, respectively. Additional time points are present for conditions where it was necessary to narrow down the point of /RNA loss. Figure 18: and RNA Recovery from Blood Stains (Swab) Exposed to Various Temperatures 84

101 Total Yield (ng) RNA RT 37 C 56 C Time The NCFS co-extraction with Nucleospin columns was used to extract and RNA from saliva stains (swab) from 3 donors incubated for various amounts of time at RT, 37 C, and 56 C. Additional time points are present for conditions where it was necessary to narrow down the point of /RNA loss. Figure 19: and RNA Recovery from Saliva Stains (Swab) Exposed to Various Temperatures 85

102 Total Yield (ng) RNA RT 37 C 56 C Time The NCFS co-extraction with Nucleospin columns was used to extract and RNA from semen stains (swab) from 3 donors incubated for various amounts of time at RT, 37 C, and 56 C. Figure 20: and RNA Recovery from Semen Stains (Swab) Exposed to Various Temperatures 86

103 Total Yield (ng) RNA RT 37 C 56 C Time The NCFS co-extraction with Nucleospin columns was used to extract and RNA from vaginal secretion swabs from 3 donors incubated for various amounts of time at RT, 37 C, and 56 C. Additional time points are present for conditions where it was necessary to narrow down the point of /RNA loss. Figure 21: and RNA Recovery from Vaginal Secretion Swabs Exposed to Various Temperatures 87

104 A % Alleles Present/Positive RNA Results B % Alleles Present/Positive RNA Results month 3 months 6 months 1 year Time (37 C) 1 month 3 months 6 months 1 year Time (56 C) RNA 1 (HBB) RNA 2 (ALAS2) RNA 1 (HBB) RNA 2 (ALAS2) Graphs show the percent of expected alleles present (- Identifiler ) and the percent of positive RNA detection results (singleplex) for the blood stains (swab) from 3 donors. Samples were incubated up to 1 year at RT (not shown), 37 C (A) and 56 C (B). Figure 22: and RNA Stability in Blood Stains (Swab) Incubated at 37 C and 56 C 88

105 A % Alleles Present/Positive RNA Results B % Alleles Presnt/Positive RNA Results month 3 months 6 months 1 year Time (37 C) 1 month 3 months 6 months 1 year Time (56 C) RNA (HTN3) RNA (HTN3) Graphs show the percent of expected alleles present (- Identifiler ) and the percent of positive RNA detection results (RNA- singleplex) for the saliva stains (swab) from 3 donors. Samples were incubated up to 1 year at RT (not shown), 37 C (A) and 56 C (B). Figure 23: and RNA Stability in Saliva Stains (Swab) Incubated at 37 C and 56 C 89

106 % Alleles Present/Positive RNA Results month 3 months 6 months 1 year Time (56 C) RNA 1 (PRM2) RNA 2 (TGM4) Graphs show the percent of expected alleles present (- Identifiler ) and the percent of positive RNA detection results (RNA- duplex) for the semen stains (swab) from 3 donors. Samples were incubated up to 1 year at 56 C. Results for the RT and 37 C conditions are not shown as no reduction in recoverability was observed at these temperatures. Figure 24: and RNA Stability in Semen Stains (Swab) Incubated at 56 C 90

107 A % Alleles Present/Positive RNA Results B % Alleles Present/Positive RNA Results month 3 months 6 months 1 year Time (37 C) 1 month 3 months 6 months 1 year Time (56 C) RNA (MUC4) RNA (MUC4) Graphs show the percent of expected alleles present (- Identifiler ) and the percent of positive RNA detection results (RNA- singleplex) for the vaginal secretion swabs from 3 donors. Samples were incubated up to 1 year at RT (not shown), 37 C (A) and 56 C (B). Figure 25: and RNA Stability in Vaginal Secretion Swabs Incubated at 37 C and 56 C 91

108 A % Alleles Present/Positive RNA Results RNA 1 (HBB) RNA 2 (ALAS2) B % Alleles Present/Positive RNA Results RNA1 (HBB) RNA 2 (ALAS2) C % Alleles Present/Positive RNA Results Time (37 C) RNA 1 (HBB) RNA 2 (ALAS2) Time (37 C) Time (37 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- singleplex) for the three blood donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 37 C condition are shown. Figure 26: and RNA Stability in Blood Stains Made on Different Substrates, 37 C 92

109 A % Alleles Present/Positive RNA Results RNA 1 (HBB) RNA 2 (ALAS2) B % Alleles Present/Positive RNA Results RNA 1 (HBB) RNA 2 (ALAS2) C % Alleles Present/Positive RNA Results Time (56 C) RNA 1 (HBB) RNA 2 (ALAS2) Time (56 C) Time (56 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- singleplex) for the three blood donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 56 C condition are shown. Figure 27: and RNA Stability in Blood Stains Made on Different Substrates, 56 C 93

110 A RNA 1 RNA 2 No detection 28/32 alleles B RNA 1 RNA 2 * 22/32 alleles 94

111 C RNA 1 RNA 2 No detection * 8/32 alleles Blood stains were made on swabs (A), carpet (B), and denim (C). The (Identifiler ) and RNA (HBB and ALAS2 singleplexes) profiles obtained from 1 donor at the 1 year time point for the 56 C condition are shown. The number of alleles and RNA marker(s) present are indicated. Asterisk (*) indicates a difference in stability from the swab samples. Figure 28: and RNA Profiles (Blood Stains on Different Substrates) 95

112 A % Alleles Present/Positive RNA Results RNA B % Alleles Present/Positive RNA Results100 RNA Time (37 C) Time (37 C) C % Alleles Present/Positive RNA Results RNA Time (37 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- singleplex) for the three saliva donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 37 C condition are shown. Figure 29: and RNA Stability in Saliva Stains Made on Different Substrates, 37 C 96

113 A % Alleles Present/Positive RNA Results RNA B % Alleles Present/Positive RNA Results100 RNA C % Alleles Present/Positive RNA Results Time (56 C) RNA Time (56 C) Time (56 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- singleplex) for the three saliva donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 56 C condition are shown. Figure 30: and RNA Stability in Saliva Stains Made on Different Substrates, 56 C 97

114 A RNA 10/32 alleles B RNA * 10/32 alleles 98

115 C RNA * 2/32 alleles Saliva stains were made on swabs (A), carpet (B), and denim (C). The (Identifiler ) and RNA (HTN3 singleplex) profiles obtained from 1 donor at the 1 year time point for the 56 C condition are shown. The number of alleles and RNA marker present are indicated. Asterisk (*) indicates a difference in stability from the swab samples. Figure 31: and RNA Profiles (Saliva Stains on Different Substrates) 99

116 A % Alleles Present/Positive RNA Resuts RNA 1 (PRM2) RNA 2 (TGM4) B % Alleles Present/Positive RNA Resuts RNA 1 (PRM2) RNA 2 (TGM4) C % Alleles Present/Positive RNA Results Time (37 C) RNA 1 (PRM2) RNA 2 (TGM4) Time (37 C) Time (37 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- duplex) for the three semen donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 37 C condition are shown. Figure 32: and RNA Stability in Semen Stains Made on Different Substrates, 37 C 100

117 A % Alleles Present/Positive RNA Results RNA 1 (PRM2) RNA 2 (TGM4) B % Alleles Present/Positive RNA Results RNA 1 (PRM2) RNA 2 (TGM4) Time (56 C) Time (56 C) C % Alleles Present/Positive RNA Results RNA 1 (PRM2) RNA 2 (TGM4) Time (56 C) Graphs show the average percent of expected alleles present (- Identifiler ) and the percent of positive detection results (RNA- duplex) for the three semen donors. Stains were made on swabs (A), carpet (B), and denim (C). The results for the 56 C condition are shown. Figure 33: and RNA Stability in Semen Stains Made on Different Substrates, 56 C 101

118 A RNA 1 and 2 Full profile B RNA 1 and 2 30/32 alleles 102

119 C RNA 1 and 2 * * 14/32 alleles Semen stains were made on swabs (A), carpet (B), and denim (C). The (Identifiler ) and RNA (PRM2 and TGM4 duplex) profiles obtained from 1 donor at the 1 year time point for the 56 C condition are shown. The number of alleles and RNA marker present are indicated. Asterisk (*) indicates a difference in stability from the swab samples. Figure 34: and RNA Profiles (Semen Stains on Different Substrates) 103

120 Total Yield (ng) RNA OS-C Time OS-UC The NCFS co-extraction with Nucleospin columns was used to extract and RNA from blood stains (from 4 donors) incubated for various amounts of time outside covered (OS-C) and uncovered (OS-UC). Real time PCR (Quantifiler Human) and Ribogreen RNA quantitation were used for and RNA quantitation, respectively. Figure 35: Recovery of and RNA from Blood Stains (Outside) 104

121 RFUs HBB ALAS OS-C Time OS-UC Average HBB and ALAS2 peak heights (for 4 donors) for the outside covered and uncovered blood stains exposed for up to 1 year. Two separate singleplexes were used for c amplification. Figure 36: Average HBB and ALAS2 Peak Heights: Blood (Outside) 105

122 A Average RFUs Time 0 1 day 3 days 1 week 4 weeks 3 months 6 months 1 year 0 B 1400 Locus 1200 Average RFUs Time 0 1 day 3 days 1 week 4 weeks 3 months 6 months 1 year Locus Average allele peak heights (RFUs) for the outside covered (A) and uncovered (B) blood stains from 4 donors exposed for up to 1 year. The AmpFlSTR Identifiler kit was used for STR amplification. Figure 37: Average Allele Peak Heights: Blood (Outside) 106

123 A % Alleles Present/Positive RNA Results B day 3 days 1 week 4 weeks 3 months 6 months 1 year Time (Covered) RNA 1 (HBB) RNA 2 (ALAS2) % Alleles Present/Positive RNA Results RNA 1 (HBB) RNA 2 (ALAS2) day 3 days 1 week 4 weeks 3 months 6 months 1 year Time (Uncovered) A comparison of the and RNA profiling success rates for the outside covered (A) and uncovered (B) blood stains. Graph shows an average (n = 4) percent of alleles present () and number of samples for which HBB and ALAS2 were detected (RNA). Figure 38: and RNA Stability in Blood Stains Exposed to Different Environmental Conditions 107

124 A RNA 1 RNA 2 Full profile B RNA 1 RNA 2 No detection Full profile 108

125 C RNA 1 * RNA 2 No detection 12/32 alleles Blood stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, and sunlight). The (Identifiler ) and RNA (HBB and ALAS2 singleplexes) profiles obtained from 1 donor at the Time 0 (A), 3 month (B), and 6 month (C) time points are shown. The number of alleles detected and RNA marker present are indicated. Asterisk (*) indicates if or RNA was recovered for a longer period of time. Figure 39: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Covered) 109

126 A RNA 1 RNA 2 Full profile B RNA 1 * RNA 2 No detection 10/32 alleles 110

127 C RNA 1 * RNA 2 No detection No profile Blood stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, sunlight, and rain). The (Identifiler ) and RNA (HBB and ALAS2 singleplexes) profiles obtained from 1 donor at the Time 0 (A), 1 week (B), and 4 week (C) time points are shown. The number of alleles and RNA marker present are indicated. Asterisk (*) indicates if or RNA was recovered for a longer period of time. Figure 40: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Uncovered) 111

128 Total Yield (ng) RNA day 3 days 1 week 4 weeks 0 1 day 3 days 1 week 4 weeks OS-C Time OS-UC The NCFS co-extraction with Nucleospin columns was used to extract and RNA from saliva stains (from 4 donors) incubated for various amounts of time outside covered (OS-C) and uncovered (OS-UC). Figure 41: Recovery of and RNA from Saliva Stains (Outside) 112

129 RFUs Covered Uncovered day 3 days 1 week 4 weeks 0 1 day 3 days 1 week 4 weeks Time Average HTN3 peak heights (for 4 donors) for the outside covered and uncovered saliva stains exposed for up to 4 weeks. A singleplex was used for c amplification. Figure 42: Average HTN3 Peak Height: Saliva (Outside) 113

130 A Average RFUS Time 0 1 day 3 days 1 week 4 weeks B 700 Locus Average RFUS Time 0 1 day 3 days 1 week 4 weeks 0 Locus Average allele peak heights (RFUs) for the outside covered (A) and uncovered (B) saliva stains from 4 donors exposed for up to 4 weeks. The AmpFlSTR Identifiler kit was used for STR amplification. Figure 43: Average Allele Peak Heights: Saliva (Outside) 114

131 A % Alleles Present/Positive RNA Results RNA B day 3 days 1 week 4 weeks Time (Covered) % Alleles Present/Positive RNA Results RNA day 3 days 1 week 4 weeks Time (Uncovered) A comparison of the and RNA profiling success rates for the outside covered (A) and uncovered (B) saliva stains. Graph shows an average (n = 4) percent of alleles present () and samples for which HTN3 was detected (RNA). Figure 44: and RNA Stability in Saliva Stains Exposed to Different Environmental Conditions 115

132 A RNA Full profile B RNA 29/32 alleles 116

133 C RNA No detection * 17/32 alleles Saliva stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, and sunlight). The (Identifiler ) and RNA (HTN3 singleplex) profiles obtained from 1 donor at the Time 0 (A), 1 week (B), and 4 week (C) time points are shown. The number of alleles and RNA marker present are indicated. Asterisk (*) indicates if or RNA was recovered for a longer period of time. Figure 45: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Covered) 117

134 A RNA Full profile B RNA Full profile 118

135 C No detection RNA 3/32 alleles Saliva stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, sunlight, and rain). The (Identifiler ) and RNA (HTN3 singleplex) profiles obtained from 1 donor at the Time 0 (A), 3 day (B), and 1 week (C) time points are shown. The number of alleles and RNA marker present are indicated. Figure 46: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Uncovered) 119

136 Total Yield (ng) RNA day 3 days 1 week 4 weeks 0 1 day 3 days 1 week 4 weeks OS-C Time OS-UC The NCFS co-extraction with Nucleospin columns was used to extract and RNA from semen stains (from 2 donors) incubated for various amounts of time outside covered (OS-C) and uncovered (OS-UC). Figure 47: Recovery of and RNA from Semen Stains (Outside) 120

137 RFUs PRM2 TGM day 3 days 1 week 4 weeks 0 1 day 3 days 1 week 4 weeks OS-C Time OS-UC Average PRM2 and TGM4 peak heights (for 2 donors) for the outside covered and uncovered semen stains exposed for up to 1 year. A duplex was used for c amplification. Figure 48: Average PRM2 and TGM4 Peak Heights: Semen (Outside) 121

138 A Average RFUS Time 0 1 day 3 days 1 week 4 weeks Locus B 1000 Average RFUs Time 0 1 day 3 days 1 week 4 weeks Locus Average allele peak heights (RFUs) for the outside covered (A) and uncovered (B) semen stains from 2 donors exposed for up to 4 weeks. The AmpFlSTR Identifiler kit was used for STR amplification. Figure 49: Average Allele Peak Heights: Semen (Outside) 122

139 A % Alleles Present/Positive RNA Results B day 3 days 1 week 4 weeks Time (Covered) RNA 1 (PRM2) RNA 2 (TGM4) % Alleles Present/Positive RNA Results RNA 1 (PRM2) RNA 2 (TGM4) day 3 days 1 week 4 weeks Time (Uncovered) A comparison of the and RNA profiling success rates for the outside covered (A) and uncovered (B) semen stains. Graph shows an average (n = 2) percent of alleles present () and samples for which PRM2 and TGM4 were detected (RNA). Figure 50: and RNA Stability in Semen Stains Exposed to Different Environmental Conditions 123

140 A RNA 1 and 2 Full profile B RNA 1 and 2 31/32 alleles 124

141 C RNA 1 and 2 * 3/32 alleles Semen stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, and sunlight). The (Identifiler ) and RNA (PRM2 and TGM4 duplex) profiles obtained from 1 donor at the Time 0 (A), 1 week (B), and 4 week (C) time points are shown. The number of alleles and RNA marker present are indicated. Asterisk (*) indicates if or RNA was recovered for a longer period of time. Figure 51: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Covered) 125

142 A RNA 1 and 2 Full profile B RNA 1 and 2 15/32 alleles 126

143 C RNA 1 and 2 Not detected No profile Semen stains were made on t-shirt cotton and incubated up to 1 year outside (exposed to heat, humidity, sunlight, and rain). The (Identifiler ) and RNA (PRM2 and TGM4 duplex) profiles obtained from 1 donor at the Time 0 (A), 3 day (B), and 1 week (C) time points are shown. The number of alleles and RNA marker present are indicated. Figure 52: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Uncovered) 127