Quality Control: Microarray
We are using three sources of RNA for our microarray
- Operating room samples from vascular, heart and
- Mouse tissues taken at the time of sacrifice
- Cell culture experiments
The mouse tissues are either pooled from genetically
in-bred animals performed in replications of at least four to provide
independent confirmation of results.
The tissue culture samples are run in replicates
of no fewer than four samples.
The operating room samples from humans are paired
with samples from non-diseased portions of the discarded specimen,
determined by immunohistochemistry, whenever such tissue is available.
RNA is purified using Trizol reagent and analyzed
on the Agilent 2100 Bioanalyzer to exclude RNA degradation.
15-20 ug of total RNA are reverse transcribed using
RnaseH negative reverse transcriptase and the resultant cDNA labeled
using fluorescent dyes conjugated to amino-allyl modified dNTP. Comparisons
of the reference sample to the treatment sample are made twice, reversing
the labeling of the reference and treatment with Cy3 and Cy5 dyes.
Performed in 5 x SSC with formamide at 42 deg C for
Washed at room temp with the most stringent wash
being 0.2 x SSC.
Read at PMTs that produce equivalent intensities
for Cy3 and Cy5 signals. Intensities are imported into an Excel spreadsheet
A quality metric is calculated as described by A.
Dudlee at http://genetics.med.harvard.edu/~dudley/oligo_arrays/calculate.html
and arrays must be in the >85% range to be considered good-quality
We are currently performing six hybridizations to
establish reproducibility. Testing is in progress to determine if
that is sufficient or excessive.
Dye reversal is used as described above
Altered expression of genes of critical interest
will be confirmed in our lab by real-time RT-PCR.
Back to Quality Control