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Affymetrix miRNA analysis Gene Microarray Shared Resource User Forum

Affymetrix miRNA analysis Gene Microarray Shared Resource User Forum. November 2009. Affymetrix miRNA array:. Implemented in GMSR, summer 2009 Probe feature size 11um; mini array format 4 identical probes per miRNA (also probes for snoRNAs)

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Affymetrix miRNA analysis Gene Microarray Shared Resource User Forum

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  1. AffymetrixmiRNA analysisGene Microarray Shared Resource User Forum November 2009

  2. AffymetrixmiRNA array: 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. AffymetrixmiRNA array content

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

  5. RNA isolation recommendations • QiagenmiRNeasy kits, Applied BiosystemsmiRVana kits, InvitrogenTrizol 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. Quality assessment procedures 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. RNA sample input for FlashTag • 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. Post-hybridization processing • 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)

  9. Partek miRNA Analysis Workflow • 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. AMC GeneChip miRNA assay cost *Price currently under review

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