Affymetrix miRNA analysis Gene Microarray Shared Resource User Forum
description
Transcript of Affymetrix miRNA analysis Gene Microarray Shared Resource User Forum
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November 2009
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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
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Organism Non-coding RNA Content
Human miRNA 847
Human snoRNA 900
Human scaRNA 22
Mouse miRNA 609
Rat miRNA 351
Canine miRNA 177
Rhesus Monkey miRNA 485
Rice (Asian) miRNA 275
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Vial 8: RNA Spike Control Oligos
Poly (A) tailed RNA
Poly (A) tail
FlashTag Ligation MixLigase
RNA3’5’
ATPPoly A Polymerase
3DNA with15 biotins
Microarray Featureor ELOSA Well
Biotin detection withStreptavidin-PE or Streptavidin-HRP
Biotin-labeled RNA
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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
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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
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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.
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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)
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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
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Service/array Cost per sampleRNA 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