Quality Control: Microarray

RNA

We are using three sources of RNA for our microarray studies:

1. Operating room samples from vascular, heart and lung tissues
2. Mouse tissues taken at the time of sacrifice
3. 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.

Hybridization

Performed in 5 x SSC with formamide at 42 deg C for 16 h.

Washed at room temp with the most stringent wash being 0.2 x SSC.

Analysis

Read at PMTs that produce equivalent intensities for Cy3 and Cy5 signals. Intensities are imported into an Excel spreadsheet for analysis.

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 analyses.

Confirmation

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.

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