November 2009
 Implemented
 Probe
in GMSR, summer 2009
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
5’
RNA
ATP
Poly A Polymerase
3’
Vial 8: RNA Spike
Control Oligos
Poly (A) tail
Poly (A) tailed RNA
3DNA with
15 biotins
FlashTag Ligation Mix
Ligase
Biotin-labeled RNA
Biotin detection with
Streptavidin-PE or
Streptavidin-HRP
Microarray Feature
or ELOSA Well



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
AMC GeneChip miRNA assay cost
Service/array
Cost 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