1. November 2009

2.  Implemented in GMSR, summer 2009  Probe feature size 11um; mini array format  4 identical probes per miRNA (also probes for snoRNAs)  46,228 probes comprising 7815 probe sets (6703 miRNA probe sets)  71 organisms represented

3. Organism Non-coding RNAContent HumanmiRNA847 HumansnoRNA900 HumanscaRNA22 MousemiRNA609 RatmiRNA351 CaninemiRNA177 Rhesus MonkeymiRNA485 Rice (Asian)miRNA275

4. Genisphere FlashTag Biotin RNA labeling assay http://www.genisphere.com/array_detection_flashtag.html Vial 8: RNA Spike Control Oligos Poly (A) tailed RNA Poly (A) tail FlashTag Ligation Mix Ligase RNA 3’5’ ATP Poly A Polymerase 3DNA with 15 biotins Microarray Feature or ELOSA Well Biotin detection with Streptavidin-PE or Streptavidin-HRP Biotin-labeled RNA

5.  Qiagen miRNeasy kits, Applied Biosystems miRVana kits, Invitrogen Trizol reagent, Marligen Vantage kits  Low molecular weight (LMW) RNA enrichment is not required but may be useful if RNA is degraded or if comparisons of mature and precursor miRNA is of interest  Elute or re-suspend RNA in nuclease-free water. Do not use a buffer that contains EDTA, which can inhibit labeling

6. RNA  Concentration measurements are required for ATP calculation  Qualitative assessments can be performed using the Agilent Bioanalyzer and the small RNA assay kit Enzyme Linked Oligosorbent Assay (ELOSA)  Two microliters of target is required  Measures labeling performance  If ELOSA fails, it could indicate the presence of inhibitors in the sample

7.  Different tissues contain different amounts of microRNA and cell lines have less miRNA compared to tissue  0.1-3µg total RNA (1µg total RNA is recommended)  LMW enriched from 0.1-3µg total RNA (300ng LMW RNA is recommended for most purification techniques). If working with limited amounts of RNA the core will need to know how much total RNA was added to the LMW enrichment procedure  Maximum assay input volume is 8µl.

8.  Inspect array image scans using AGCC (Command Console) Viewer  Core performs basic QA using Affymetrix miRNA QC tool  Downstream analysis performed by User/Data analyst using and third party/custom software. One example of GMSR User-available software that is compatible with miRNA data is Partek Genomics Suite. (www.partek.com)www.partek.com

9.  Principal Components Analysis (PCA) for exploratory data analysis and to identify any major outliers  Define experimental factors and run ANOVA analysis to generate “results list” of differentially expressed miRNAs  Filter results list by statistical p-value and/or other criteria  If desired, create a species-specific filter to eliminate those species from list that are not of interest  Combine/correlate miRNAs with their mRNA targets

10. Service/arrayCost per sample RNA assessment$8 RNA processing fee$35 ( for each group of 10 samples ) RNA labeling$90 miRNA array$195 Hybridization & Data prep$120* Data management$50* Total$463 per sample *Price currently under review AMC GeneChip miRNA assay cost