November 2009

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

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

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

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

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

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.

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)

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

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