BIOO RESEARCH PRODUCTS. ALL-TAIL Kit Manual For Extreme 3 RACE Catalog #: 5205

BIOO RESEARCH PRODUCTS ALL-TAIL Kit Manual For Extreme 3 RACE Catalog #: 5205 BIOO Scientific Corp. 2010

TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Procedure Overview... 2 Kit Contents, Storage and Shelf Life... 3 Required Materials Not Provided With the Kit... 3 ALL-TAIL KIT PROTOCOL... 4 Ligation of Linker to RNA... 4 Reverse Transcription... 4 PCR Amplification... 5 Sizing or Sequencing... 6 ADDITIONAL RESOURCES... 7 Preparing and Running Polyacrylamide Gels... 7 TROUBLESHOOTING... 9 Analysis of Alternative Polyadenylation Sites... 9 Poly(A) Signal for Target Gene Not Detected... 9 Verify the Amplified Sequence is a Poly(A) Tail... 9 No Signal or Weak Signal... 10 More than one band after PCR amplification... 10 The ALL-TAIL Kit is intended for laboratory use only. This product is NOT for clinical diagnostic use. ALL-TAIL is a trademark of Bioo Scientific Corporation (BIOO).

GENERAL INFORMATION GENERAL INFORMATION Product Description ALL-TAIL Kit Manual - 5205 The patent pending technology of the ALL-TAIL (ATP-independent Linker Ligation Poly(A) Tail) Kit for extreme 3 RACE, can be used to investigate the dynamic changes in poly(a) tail length that take place during gene expression, as well as the changes in 3 untranslated regions caused by differential poly(a) tail addition site selection in development, differentiation and cancer. This kit enables the precise measurement of 3 poly(a) tail length along with the ability to identify and characterize the 3 end of RNA transcripts. It can also be used to detect mirna molecules using a linker ligation technique followed by PCR. Most eukaryotic mrnas contain a poly(a) tail at their 3 end as a result of a cleavage/polyadenylation process that occurs co-transcriptionally in the nucleus. During transcription termination in eukaryotes, pre-mrnas are polyadenylated by cleaving 10-35 nucleotides after the polyadenylation signal (AAUAAA). Poly(A) polymerase then adds the poly(a) tail to the cleaved RNA end. The poly(a) tail plays an important role in a variety of cell processes, including export, translation, mrna stability and mirna regulation. The site of cleavage and poly(a) addition can vary significantly during cell growth and differentiation, leading to drastic changes in the length of the 3 untranslated region of RNAs (UTR) that may alter the ability of the transcript to be regulated by mirnas or RNA binding proteins (http://polya.umdnj.edu/polyadb/) or (http://exon.umdnj.edu/polya_svm/). The length of the poly(a) tail of mrna is variable and influences the fate of the transcript in terms of its stability and ability to be translated. The poly(a) tail and the 3 UTR may serve as an adjustable regulator of gene expression that can coordinate expression among functional classes of transcripts as well as a means of quickly adjusting cellular gene expression to address environmental changes. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 1

Procedure Overview ALL-TAIL Kit Manual - 5205 Bioo Scientific s ALL-TAIL Kit provides a sensitive, accurate and highly reproducible assay to measure the poly(a) tail length and the 3 UTR sequence of any RNA species. First, AIR Ligase (a highly efficient truncated T4 RNA ligase 2) efficiently joins an adenylated adaptor oligonucleotide to the 3 end of the total RNAs. Next reverse transcription is performed using a primer specific to the adenylated adaptor to initiate the production of cdna. Then a primer designed near the 3 end of the transcript of interest is used along with the primer specific to the adenylated adaptor to perform PCR amplification of the poly(a) tail and a short stretch of the 3 UTR. This step may be modified to amplify specific mirna sequences. Finally, the PCR products are separated on an acrylamide gel and visualized using either SYBR Gold Nucleic Acid Stain or ethidium bromide. 5 AAAAAAAAAAAAAAA rapp ddc Step 1: Ligation 5 AAAAAAAAAAAAAAA ddc Step 2: Reverse Transcriptase 5 AAAAAAAAAAAAAAA ddc 3 3 TTTTTTTTTTTTTTTT 5 Step 3: PCR 5 AAAAAAAAAAAAAAA ddc 3 3 TTTTTTTTTTTTTTTT 5 Run on non-denaturing gel BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 2

ALL-TAIL Kit Manual - 5205 Kit Contents, Storage and Shelf Life The ALL-TAIL Kit can be used to perform 20 ligation reactions, 30 reverse transcription reactions and 30 PCR reactions (50 L). Store the Control A549 Total RNA at -80 C and the rest of the kit at - 20 C. Return any unused components to their proper storage temperature. The shelf life of this kit is 6 months. Check the label on the kit box for the specific expiration date. Kit Contents Amount Storage AIR Adenylated Linker C (10 µm) 5 rapptttaaccgcgaattccag/3ddc/3 90 µl -20 C AIR Ligase 20 µl -20 C 10X AIR Ligase Buffer 40 µl -20 C Control A549 Total RNA (0.5 µg/µl) 10 µl -80 C M-MuLV Reverse Transcriptase 30 µl -20 C 10X M-MuLV buffer 60 µl -20 C 20 mm dntp mix 30 µl -20 C Linker C Universal RT-PCR Primer (50 µm) 5 -CTGGAATTCGCGGTTAAA-3 T m 50.3 o C 60 µl -20 C DuroTaq 5X Master Mix 300 µl -20 C GAPDH tail PCR primer (50 µm) 5 -CATGTAGACCCCTTGAAG-3 T m 49.5 o C 20 µl -20 C RNase-free H 2 O 2 ml RT Required Materials Not Provided With the Kit Specific PCR primer designed to the gene of interest Thermal cycler Precision pipettes Microcentrifuge Nuclease-free microcentrifuge tubes RNase-free filter tips Appropriate PCR plates or tubes for instrument Gel electrophoresis reagents and equipment Personal protective equipment BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 3

ALL-TAIL KIT PROTOCOL ALL-TAIL KIT PROTOCOL Ligation of Linker to RNA ALL-TAIL Kit Manual - 5205 To dramatically increase the efficiency, specificity and reproducibility of this step, we developed a patent pending technique in which an oligomer used in the ligation contains an ATP moiety at its 5 end (referred to as an adenylated linker). This allows the ligation reaction to be performed in the absence of added ATP in the reaction and ensures that the only polynucleotide able to ligate in the reaction will be the adenylated oligo to the 3 OH group at the end of endogenous cellular RNAs. In addition, the adenylated linker is blocked at its 3 end with a dideoxycytidine. The sequence of the AIR Adenylated Linker C supplied in the kit is shown above. AIR Adenylated Linker C is also offered as a stand-alone product from Bioo Scientific (Cat # 510205). Custom linkers containing different melting temperatures are available upon request. 1. Isolate total RNA or enriched mirna from sample. We recommend using BiooPure (Bioo Scientific Cat # 5301-02) for the isolation of total or enriched small RNA. 2. Thaw frozen reagents on ice and mix completely by gently flicking the tube. Centrifuge briefly to ensure all contents are at the bottom of the tube. Add the reagents as shown in the table below to a nuclease-free tube. (**optional: include 0.5ul of RNase Inhibitor into the ligation reaction**) 3. Mix by gently flicking the tube, briefly spin the tube and incubate at 22 C for 1 hour using a temperature controlled incubator. RNA Ligation Reaction: Reagent AIR Adenylated Linker C (10 M) 5 rapptttaaccgcgaattccag/3ddc/3 10X AIR Ligase Buffer AIR Ligase Total RNA (1 g) RNase-free water (BTV 20uL total vol.) Amount 4.5 L 2 L 1 L x L to 20 L 4. Samples can be used directly for Reverse Transcription or stored at -20 C until you are ready to proceed. Reverse Transcription Note: The protocol below applies to a single 20 L reverse transcription reaction. Master mixes for multiple reactions can be made by increasing the volumes of reaction components. In this step, Linker C Universal RT-PCR Primer, a primer complementary to the AIR Adenylated Linker C, is mixed with the ligation reaction from the Ligation of Linker to RNA. The mixture is treated with M-MuLV Reverse Transcriptase to generate cdna. 1. Thaw frozen reagents on ice and mix completely by gently flicking the tube. Centrifuge briefly to ensure all contents are at the bottom of the tube. 2. Add the reagents as shown in the table below into a nuclease-free tube. Mix gently, briefly spin the tube contents and place on ice. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 4

ALL-TAIL Kit Manual - 5205 Reverse Transcription Reaction Mix: 3. Incubate at 42 C for 30 minutes and then 92 C for 5 minutes. 4. Samples can be used directly for PCR Amplification or stored at -20 C until ready to proceed. PCR Amplification Use the following protocol to amplify the cdna using PCR. The following protocol applies to a single 50 L PCR reaction: 1. Thaw frozen reagents on ice and mix by gently flicking the tube. Centrifuge briefly to ensure all contents are at the bottom of the tube. 2. Add the reagents as shown in the table below to a nuclease-free tube. Add sufficient RNase-free water to bring the final reaction volume to 50 L. Mix gently and briefly spin the tube contents. PCR Mix Reagent Amount RNase-free water 11 L RNA ligation reaction from Step 1 4 L 10X M-MuLV buffer 2 L M-MuLV Reverse Transcriptase 1 L Linker C Universal RT-PCR Primer (50 µm) 1 L 20 mm dntp mix 1 L Reagent RNase-free water DuroTaq 5X PCR Master Mix GAPDH tail PCR primer or gene-specific Tail PCR primer Linker C Universal RT-PCR Primer (50 μm) 5 -CTGGAATTCGCGGTTAAA-3 T m 50.3 C cdna template from RT Reaction Amount 35.5 L 10 L 3. Place the mixture into the thermal cycler and perform a PCR reaction. We typically use the following temperature cycles for analyzing GAPDH poly(a) tail length: 1 L 1 L 2.5 L a. 1 cycle (95 C for 3 minutes) b. 30 cycles (94 C for 15 seconds, 50 C for 15 seconds and 72 C for 30 seconds) c. 1 cycle (72 C for 5 minutes) BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 5

ALL-TAIL Kit Manual - 5205 Sizing or Sequencing The size of the poly(a) tail (or 3 -end) can be analyzed by running 35ul of the reaction on a nondenaturing 10% polyacrylamide gel. Run the samples along with a ladder that contains DNA fragments that range from 10 to 1000 bp. We recommend Bioo Scientific s MW Marker (Plasmid Digest) Ready-to-Load that extends from 105 bp to 995 bp (Bioo Scientific Cat # 371001). To make a 10% acrylamide gel, mix 3 ml of 10X TBE, 10 ml of 30% acrylamide gel mix (19:1 acrylamide: bisacrylamide), 17 ml of nuclease-free water and 1.5 mg of Ammonium persulfate (APS). Add 10 l of TEMED and immediately pour into the gel apparatus and insert a comb. The figure below shows the PCR products generated from total RNA isolated from A549 cells treated with oligo(dt) and RNase H to determine the precise length of the poly(a) tail. A549 total RNA was used to measure the tail length of the GAPDH mrna. A549 A549 + Oligo dt and RNase H LADDER A100 bp 250bp 200bp 150bp 100bp 75bp 50bp A0 bp 25bp PCR products can be cloned into suitable plasmid vectors using standard techniques. The AIR Adenylated Linker C sequence 5 -TTTAACCGCGAATTCCAG-3 contains an EcoR1 site (GAATTC) to facilitate cloning and sequencing. PCR products amplified with a gene specific primer that has a restriction site can be cloned into an appropriately digested plasmid vector using standard cloning techniques. Alternatively, a quick PCR cloning kit can be used to clone the PCR products without using restriction digest. The PCR products may be used on next generation sequencing instruments with sufficient read lengths. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 6

ADDITIONAL RESOURCES ADDITIONAL RESOURCES ALL-TAIL Kit Manual - 5205 Linker C Universal RT-PCR Primer is the primer used for reverse transcription and acts as the downstream primer for the PCR reaction. The gene specific PCR primer design is an important aspect of the ability to identify the length of the poly(a) tail. We typically design primers between 50-300 nucleotides upstream of the poly(a) start site to allow for effecicent resolution of the PCR products by gel electrophoresis. We highly recommend the following databases that have algorithms for predicting poly(a) site selection (http://polya.umdnj.edu/polyadb/ and http://exon.umdnj.edu/polya_svm/). For mirna PCR primer selection please use the Sanger mirna database for primer design (http://www.mirbase.org/). For best results follow these guidelines: 1. Primers should range in length from 19 to 30 nucleotides 2. G+C content in the range of 30 to 50% 3. T m values ranging from 48-60 C 4. Analyze for cross-reactivity in the organism s database. Due to the AT-rich content in the 3 UTR sequence, it may be difficult in some cases to design an upstream primer that fits these specifications. If a primer with a T m of 48-60 C T m cannot be easily identified in your sequence, Bioo Scientific can manufacture an adenylated linker to match the T m specifications of your upstream primer. For more information about Bioo Scientific s custom AIR Adenylated Linkers email nextgen@biooscientific.com. The control GAPDH tail PCR primer is included to ensure that the PCR reaction is working effectively. In all cell lines that we have analyzed thus far the GAPDH poly(a) tails are nearly identical in size. Therefore, this makes for a nice control of RNA quality and reaction efficiency. Preparing and Running Polyacrylamide Gels 1. Choose a vertical gel electrophoresis apparatus with a well capacity of 20 μl or greater. Follow the manufacturer s instructions when assembling the gel apparatus. 2. One 10 cm x 15 cm x 1 mm gel requires 15 ml of a gel solution. To make a 10% acrylamide gel, mix 1.5 ml of 10X TBE, 5 ml of 30% acrylamide gel mix (19:1 acrylamide: bis-acrylamide), 8.5 ml of nuclease-free water and 15 mg of APS (Sigma Cat # A3678). Add 10 L of Tetramethylethylenediamine (TEMED) to the mixture, immediately pour into the gel apparatus and place the comb into the apparatus to form the wells. 3. Let sit at room temperature for at least 30 minutes for the gel to solidify. 4. Prepare the samples by diluting a gel loading buffer to 1X in the sample. We recommend either of Bioo Scientific s gel loading dyes, the 8X loading dye (Bioo Scientific Cat # 370901) or the 6X loading dye (Bioo Scientific Cat # 370801). 5. Mix and quick spin the samples to collect the tube contents at the bottom of the tube. 6. Load 20 L of the sample into each well. For each gel, load a DNA marker so that the length of the samples can be identified. 7. After the gel has run to completion, separate the plates and stain the gel in 1X TBE containing SYBR Gold Nucleic Acid Stain or ethidium bromide. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 7

10X TBE Buffer To make 2 L of 10X TBE: ALL-TAIL Kit Manual - 5205 1. Add 1400 ml of deionized water to clean 2 L capacity glass bottle. 2. Add 216 g of Tris Base. 3. Add 110 g of Boric Acid. 4. Add a clean stir bar and mix well with a stir plate. Intermittently, shake the bottle vigorously to help dissolve the solids faster. 5. Add 14.9 g of EDTA. 6. Mix on a stir plate until completely dissolved. 7. Transfer solution to a clean measuring cylinder without the stir bar. 8. Bring to volume of 2000 ml (2 L) using deionized water. 9. Transfer solution back into 2 L bottle. 10. Autoclave for 40 minutes. 11. Store at room temperature. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 8

TROUBLESHOOTING TROUBLESHOOTING ALL-TAIL Kit Manual - 5205 The most significant variable on the outcome of the experiment is the optimization of the PCR annealing temperatures. To optimize the reaction conditions for optimal annealing temperatures, we have designed linker primers with numerous T m s between 50-60 C. To obtain the list of these linker sequences please inquire to techsupport3@biooscientific.com. Analysis of Alternative Polyadenylation Sites If nested PCR produces several bands, this may indicate alternative polyadenylation cleavage sites. The potential for alternative poly(a) addition sites can be examined using the databases (http://polya.umdnj.edu/polyadb/) or (http://exon.umdnj.edu/polya_svm/). Up to 50% of genes are differentially processed at the 3 ends. This could generate significant changes in the 3 UTR sequences which often contain highly important regulatory sequences. Poly(A) Signal for Target Gene Not Detected Causes for this include the reagents were used in the wrong order, a step was skipped, the reagents are expired, the PCR machine was not set correctly or the RNA sample input was degraded. If this occurs perform a control reaction using the provided GAPDH tail PCR primer with Control A549 Total RNA. This should yield a PCR product as depicted in the gel shown above. If a product is not generated, contact technical support at 512-707-8993 or techsupport3@biooscientific.com. Verify the Amplified Sequence is a Poly(A) Tail To verify the PCR product as a poly(a) tail, we recommend performing either oligo(dt) RNase H reaction which will degrade the poly(a) tails or actinomycin D treatment. 1. To make the poly(a) minus control perform a reaction with oligo(dt) and RNase H (Ambion Cat # AM2292). In a RNase-free tube using RNase-free reagents 18 base oligo(dt) primer is mixed with total RNA in 10-fold molar excess. The reaction is heated to 95 C and slowly cooled to room temperature in a total volume of 20 μl. RNase H and 10X RNase H reaction buffer (200 mm Tris ph 8.0, 40 mm MgCl 2, 10mM DTT and 500 mm NaCl) is added and is incubated at 37 C for one hour. The sample is then phenol/chloroform extracted, precipitated and suspended in 50 µl of nuclease-free water. The concentration of the suspended RNA is determined and used in an ALL-TAIL kit reaction as an (A) 0 control. 2. Actinomycin D (Sigma Cat # A1410) treatments of cell cultures can be used. After the addition of actinomycin D at 6 g/ml final concentration, the cells are incubated for different time points (ranging from 1 hour to 24 hours). During the incubation of the cells with actinomycin D, transcription is shut down and the RNAs poly(a) tail is removed.this reduction in poly(a) tail length may be detected in this assay depending on the RNAs half life. Following the treatment of the cells, the RNA is isolated and used directly in the ALL-TAIL kit. BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 9

ALL-TAIL Kit Manual - 5205 No Signal or Weak Signal Possible Causes Gene specific PCR primers were not designed correctly PCR primer melting temperature is not compatible with linker primer melting temperature Reagents were expired or mixed with a different lot number or were prepared incorrectly. Incubation times were too short. Excessive kit stress has occurred. Low abundance mrnas being detected Recommended Action Double check the primer is the correct sequence and orientation for amplification of the poly (A) tail. Design a new primer in a different region of the UTR which enables it to be more compatible or contact Bioo Scientific about obtaining an adenylated linker with a compatible melting temperature Verify the expiration dates and lot numbers and that the reagents were prepared correctly. Time each plate separately to ensure accurate incubation times, follow protocol. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. Increase the number of PCR cycles from 30 to 50 More than one band after PCR amplification Possible Causes The PCR annealing temperature is not stringent enough or the amplicon has been over-amplified The gene of interest is differentially polyadenylated Recommended Action Test a series of gene specific primers, melting temperatures and cycles during PCR amplification. A. Check the databases to see if your gene may have a predicted poly(a) addition site: (http://polya.umdnj.edu/polyadb/) or (http://exon.umdnj.edu/polya_svm/) B. Ensure the bands are specific to the gene of interest by either cloning the PCR products into cloning vector or gel purifying and sequencing them directly. Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX 78744 USA Tel: 1.888.208.2246 Fax: (512) 707-8122 Made in USA BIOO Research Products Group info@biooscientific.com techsupport3@biooscientific.com www.biooscientific.com BIOO RESEARCH PRODUCTS WWW.BIOOSCIENTIFIC.COM 10