All figures and Charts are attached in the Appendix (Section 1. Lymphoma Genomic Targets - Pathology).

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1 Progress Summary by Experimental Arm 1. Lymphoma Genomic Targets (Pathology) beta-actin (ACTB), TATA box binding protein (TBP) 2. Infectious Disease and Human Genome Genomic Targets (IDRC) meca gene, drug resistance in MRSA (methicillin-resistant Staphylococcus aureus); tolllike receptor 4 (TLR4) 1. Department of Pathology, Progress (PATH) Primary Outcome Measures: a) DNA and RNA concentration, and b) Real-time PCR Cycle number (a surrogate for stored DNA concentration) Secondary outcomes: Measures of DNA and RNA purity, A260/280 ratio and A260/230 ratio Controls: Baseline determination of fresh samples without storage All figures and Charts are attached in the Appendix (Section 1. Lymphoma Genomic Targets - Pathology). Prior to performing our actual tests, Crystal Loftin, undergraduate student, was trained on the technique of DNA and RNA isolations using the Qiagen, All-Prep DNA and RNA isolation kit. The DNA and RNA were isolated from three samples of frozen lymphoma cells (patient samples). The concentration and purity were measured using the NanoDrop system. The practice samples were then used to optimize the primers for our real time PCR experiments. This optimization took a fair amount of time, resulting in a delay of testing the designated lymphoma samples. The reagent kits used were the iq Sybr Green Supermix for DNA, and the iscript One-Step RT- PCR kit with Sybr Green for RNA (both kits from BIO-RAD). The two primers used for the experiments β-actin (ACTB) and TATA-box protein 2 (TBP). Both primer sets were generated by Invitrogen. In order to start in a timely manner, Crystal isolated DNA and RNA from the lymphoma samples, and real-time PCR was performed on those samples with expert help. Chart 1 (Appendix) displays the concentration and A260/280 for the baseline samples. After the measurements were made, each sample was divided into two sets: 5ul aliquots were placed into 0.5ml tubes, then placed into the -80 o C freezer; 5ul aliquost were placed into labeled wells of the Biomatrica 96-well DNA-stable and 96-well RNA-stable plates, respectively. The plates were stored in a humidity free cabinet supplied by Biomatrica. Real time PCR was performed on these samples to obtain a baseline C t value. In Fig. 1 (Appendix), please note the first image is DNA, the second RNA. At the three month time point, Crystal alone performed the Real Time PCR on both the DNA and RNA. Chart 2 and Figure 2 represents the 3 month storage comparison of the DNA and Chart 3 and Figure 3 (Appendix) is the 3 month comparison of the RNA. RNA is highly unstable; therefore its long term storage is extremely important. As we continue our work, Crystal will isolate additional DNA and RNA lymphoma samples obtain baseline data in order to optimize our dry storage technique of RNA. Our studies so far have shown a fair

2 comparison of the dry storage v. -80 o C freezer. We will perform our 6 month evaluation in April The Infectious Disease Research Core (IDRC) Progress Primary Outcome Measures: a) DNA concentration, and b) Real-time PCR Cycle number (a surrogate for stored DNA concentration) Secondary outcomes: Measures of DNA purity, A260/280 ratio and A260/230 ratio Controls: Colony forming units/ml of bacteria placed into storage to document bacterial density and its relationship to DNA concentration All Figures and tables are attached in the Appendix (Section 2. Infectious Disease and Human Genome Genomic Targets -IDRC) Summary of progress towards stated project impacts and outcomes, and how progress is tracking with the timeline provided in your original proposal. Green Grant IDRC Project Progress Summary and Timeline Phase 1: Project Startup 1. Reagent and Materials 1.1. Primers and probes ordered and diluted to working stock dilution 1.2. BioMatrica DNAStable and other materials/reagents ordered and prepared 1.3. Reagent aliquots were pre-prepared to accommodate each time point planned for extraction, rehydration of dry stored nucleic acids and real-time polymerase chain reaction 2. Documentation and Procedures 2.1. Standard operating procedures (SOPs) and flow diagrams detailing experimental design (Figure 4) were written by EI or revised to accommodate Green Grant objectives. Six procedures were written or adapted: Cultivation of Bacteria, DNA Extraction, Nucleic Acid Quantification and Purity Measurements, Serial Dilution of Bacteria, Bacterial Colony Plate Counts, and Calculations for Dilutions and Colony Forming Units(CFU)/mL Calculations for bacterial densities and primer/probe calculations were made 3. Data Entry and Statistical Analysis Plan

3 3.1. Data entry log designed in conjunction with biostatistician at Infectious Disease Research Core 3.2. Statistical Plan Created: In order to determine whether a difference exists in storage method over time by Ct values of matched densities a General Linear Model (GLM) will be employed. Storage method and time intervals will be considered fixed effects in the model and an interaction between the two variables of interest will be determined by an F-test at a level of significance of α = No assumptions will be made regarding the underlying distribution however log transformations may be employed if necessary to meet model assumptions more closely. Statistical analysis will be performed in SPSS 19 (Chicago, Illinois, USA). 4. Training and Student Mentorship 4.1. Weekly or bi-monthly meetings with Dr. Wolk, IDRC manager, and student (EI) 4.2. EI was trained to perform all techniques listed as SOPs EI was trained to operate the Eppendorf MasterCycler ep for real-time PCR assay to document DNA stability 4.4. EI was trained to perform bacterial density plate counts to document CFU/mL for bacteria used in experiments 4.5. EI was trained to extract DNA on the Qiagen EZ-1 Biorobot, Tissue Kit with Bacterial Card EI was trained to perform NanoDrop analysis to determine nucleic acid concentrations Phase 2: Student Dry Runs 1. Student Testing Experience 1.1. DNA Extraction and PCR dry runs were performed according to SOPs DNA was quantified by NanoDrop Technology. 2. Results 2.1. DNA Concentration results showed lower than anticipated DNA concentrations in NanoDrop data and poor amplification was observed in real-time PCR data for the SA and meca targets. This data was supported by lower than anticipated colony counts (Table 1) Upon investigation EI and DW discovered that an old agar subculture had been used. Colonies were used from a 2-week old, refrigerated plate. Therefore, SOP and training was revised to highlight the importance of fresh subcultures and repeat testing was initiated. 3. Conclusion and Action Plan 3.1. Experiments did not use fresh agar plate subcultures of bacteria. Repeat testing was necessary Phase 3: Assay Optimization for DNA Extraction and PCR 1. Student Testing Experience 1.1. Fresh subculture were used 1.2. DNA Extraction and PCR dry runs were performed according to SOPs Colony counts were performed and characterized (Table 2)

4 1.4. DNA was quantified by NanoDrop Technology. (Table 3) 2. Results and Conclusions This data still showed lower than anticipated colony counts (Table 2); however, DNA Concentration results showed acceptable yields of DNA concentrations and in purity in NanoDrop data (Table 3). Poor amplification was observed in real-time PCR data for the SA and meca targets (Figure 4). 3. Actions /23/2011, Protocol optimization based on previous results: Changed Extraction protocol from FRESH subcultures and optimized for add 2-3 colonies directly in G2 buffer (buffer from EZ- 1 pre-extraction protocol), heated sample for 95 C (instead of 60C) to increase DNA yield) and then placed them on the extractor after the heat step Results: NanoDrop results were improved but cycler stopped reaction at 28/40 cycles for unknown reason. Conclusion: Error with the Eppendorf Mastercycler. Called technical support, performed reboot of both laptop and cycler /28-12/5/2011, Protocol: Repeat of the above experiment due to instrument error. Results: NanoDrop- results were acceptable. Results: While thermal cycler being evaluated, performed gel electrophoresis of the PCR amplicon and there was no amplified DNA in the sample. NanoDrop results below depict acceptable concentration and purity of DNA from optimized extraction and that concentration increases as bacterial density increases. Instrument manufacturer contacted for service. Plan to use alternate thermal cycler for next evaluation 4. 12/5/11-1/15/2012: Finals and Holiday closure 4.1. Request for thermal cycler service delayed over holiday. Alternate PCR cycler will be tested while repairs take place of Mastercycler ep while in repair. Repeat runs planned on alternate cycer,2/11/2012, 4.2. Once cycler fixed: At the rehydration points and thawing points (3m, 6m, 12m, 2yrs, etc ) the DNA will be analyzed again to determine which long term storage method better preserves DNA. The IDRC arm is slightly behind the Pathology arm due because primers and probes were not pre-validated for this arm.

5 Appendix: Supporting Data 3. Lymphoma Genomic Targets (Pathology) Chart 1: displays the concentration and A260/280 for the baseline samples Sample Date ng/ul A260 A280 A260/280 WC-RNA 10/10/ SK-RNA 10/10/ LS-RNA 10/10/ WC-DNA 10/10/ SK-DNA 10/10/ LS-DNA 10/10/ Figure 1: Real time PCR was performed on these samples to obtain a baseline C t value. Note that the first image is DNA, the second RNA.

6 Chart 2: shows the data for the 3 month storage comparison of the DNA. Sample Storage Date ng/ul A260 A280 A260/280 WC-DNA dry 1/11/ SK-DNA dry 1/11/ LS-DNA dry 1/11/ WC-DNA -80 o C 1/11/ SK-DNA -80 o C 1/11/ LS-DNA -80 o C 1/11/ Figure 2: 3 month storage comparison of the DNA for each target

7 Chart C: 3 month comparison of the RNA. Sample Storage Date ng/ul A260 A280 A260/280 WC-RNA dry 1/13/ SK-RNA dry 1/13/ LS-RNA dry 1/13/ WC-RNA -80 o C 1/13/ SK-RNA -80 o C 1/13/ LS-RNA -80 o C 1/13/ Figure 3: 3 month comparison of the RNA for each target.

8 4. Infectious Disease and Human Genome Genomic Targets (IDRC) Table 1: Plate counts for meca validation dry runs (in triplicate for 3 different dilutions). Plate counts exceed acceptable difference from bacterial target density, thus DNA concentration was below the PCR limit of detection for 3 of 3 dilutions. Repeat testing required. Run Plate Colony Count Target Density Actual Density McFarland Date duplicate (50uL plated) (CFU/mL) (CFU/mL) Target 1 11/10/ ^ /10/ ^ /10/ ^ /10/ ^ /10/ ^ /10/ ^ /10/ ^ /10/ ^ /10/ ^ Table 2: Plate count Distributions Target Density (CFU/mL) for 3 different dilutions [1.5^3, 1.5^4, and 1.5^5 CFU/mL (n=3 each)], the 1.5^5 CFU/mL dilution had colony counts too numerous to count by eye. Colony Counts of dry runs were lower than expected but DNA yield was acceptable for storage and PCR (Table 3). Repeat dry runs are pending instrument repair. Distributions Target Density (CFU/mL)=1.5^3 Distributions Target Density (CFU/mL)=1.5^4 Mean Std Dev Std Err Mean Upper 95% Mean Lower 95% Difference from target = 627 CFU/ml = 2.8log Mean Std Dev Std Err Mean Upper 95% Mean Lower 95% Difference = 3.9log Table 3: DNA Concentration (n=6) derived from EZ-1 extraction of fresh bacterial colonies reveals abundant DNA in quantity and purity Inoculum Date DNA Concentration (ng/ul) A260/280 Purity ratio A260/230 Purity ratio SA 3 Large Colonies 11/28/ SA 2 Large Colonies 12/5/

9 Figure 4