An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA.

ABSTRACT:

Background

Validation of microarrays data by quantitative real-time PCR (qPCR) is often limited by the low amount of available RNA. This raised the possibility to perform validation experiments on the amplified amino allyl labeled RNA (AA-aRNA) leftover from microarrays. To test this possibility, we used an ongoing study of our laboratory aiming at identifying new biomarkers of graft rejection by the transcriptomic analysis of blood cells from brain-dead organ donors.

Results

qPCR for ACTB performed on AA-aRNA from 15 donors provided Cq values 8 cycles higher than when original RNA was used (P < 0.001), suggesting a strong inhibition of qPCR performed on AA-aRNA. When expression levels of 5 other genes were measured in AA-aRNA generated from a universal reference RNA, qPCR s

SUBMITTER: Jeanty C 

PROVIDER: S-EPMC3091691 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature