Amplification and amino allyl labeling decrease qPCR efficiency and sensitivity. One μg RNA and 100 ng AA-aRNA from human universal reference were reverse transcribed with SuperScript II. (A) Ten-fold dilutions of resulting cDNAs were subjected to qPCR using primer pairs specific for VEGFB. The amplification chart shows higher Cq values for AA-aRNA compared with RNA. (B) cDNAs obtained from reverse transcription of RNA and AA-aRNA were serially diluted and qPCR was performed to evaluate qPCR efficiency using VEGFB-specific primers. qPCR efficiency calculated from standard curves (E = [10-1/slope]-1) is down-regulated by amplification and amino allyl labeling.
Jeanty et al. BMC Genomics 2010 11:542 doi:10.1186/1471-2164-11-542