Background: RNA interference (RNAi) has been one of the most rapidly expanding areas of biological research in the past decade, revolutionizing the ability to analyze gene function. Thorough validation of siRNA duplexes is required prior to use in experimental systems, ideally by western blotting to show a reduction in protein levels. However, in many cases good antibodies are not available, and researchers must rely on RT-qPCR to detect knockdown of the mRNA species.
Findings: We have observed a phenomenon that gives a disparity between analyzing small interfering RNA (siRNA) efficacy by western blotting of the protein levels and real-time quantitative PCR (RT-qPCR) measurement of mRNA levels. Detection of this phenomenon was dependent upon the location of the target amplicon for PCR primers within the mRNA.
Conclusions: Our data suggests that for certain mRNAs, degradation of the 3' mRNA fragment resulting from siRNA mediated cleavage is blocked, leaving an mRNA fragment that can act as a template for cDNA synthesis, giving rise to false negative results and the rejection of a valid siRNA duplex. We show that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment.