%0 Journal Article
%T Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design
%A Katherine Holmes
%A Catrin M Williams
%A Elinor A Chapman
%A Michael J Cross
%J BMC Research Notes
%D 2010
%I BioMed Central
%R 10.1186/1756-0500-3-53
%X 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.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.RNA interference (RNAi) was first observed in Caenorhabditis elegans by Fire, Mello et al. [1], who found that introduction of double stranded RNA resulted in the silencing of gene expression. In the past decade RNAi has been one of the most rapidly expanding areas of biological research, allowing the development of RNAi as a therapeutic approach in the treatment of several disorders, including cancer and autoimmune disorders [2]. The RNAi pathway has also been utilized in vitro, enabling the knockdown of genes, and revolutionizing the ability to analyze gene function. The initial stage of analysis of gene function is to fully characterize the extent of siRNA mediated gene knockdown, as in many cases the gene expression is not completely inhibited. Knockdown of the mRNA is easily quantified using real-time quantitative PCR (RT-qPCR), whilst knockdown of the protein is visualized with SDS-PAGE and western blotting. Here we report upon a phenomenon which affects RT-qPCR quantification of gene knockdown, and which could result in false negative results, and the rejection of valid siRNA duplexes.RNAi is initiated by the presence of long dsRNA molecules in the cell. These are cleaved into small-interfering RNA (siRNA) duplexes, 21-26 nucleotide
%U http://www.biomedcentral.com/1756-0500/3/53