Figure 1From: An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNAAmplification and amino allyl labeling inhibit qPCR. Total RNA extracted from whole blood cells of 15 healthy brain-dead organ donors was subjected to amplification by T7 polymerase and amino allyl labeling to generate AA-aRNA. 1 μg of un-amplified and un-labeled RNA and 100 ng AA-aRNA (amplified and amino allyl labeled RNA but not coupled with fluorescent dye) were reverse transcribed using SuperScript II, resulting cDNAs were diluted 10-fold and subjected to qPCR using primers specific for ACTB. (A) Cq obtained from the 15 samples are represented in a box plot. A statistically significant difference was detected between RNA and AA-aRNA (t = -25.23; P < 0.001; paired t-test). (B) Cq obtained with RNA and AA-aRNA for each of the 15 patients were strongly correlated (R2 = 0.85; P = 0.00006; Pearson product moment correlation). Similar results were obtained in two independent experiments.Back to article page