Amplification 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.
Jeanty et al. BMC Genomics 2010 11:542 doi:10.1186/1471-2164-11-542