What are the critical factors for reliable RNAi validation using qRT-PCR?
The SABiosciences R&D team has carried out an extensive study aimed at answering this question. A thorough evaluation of the major contributing factors essential to RNAi validation using qRT-PCR was carried out. A complete description of this study can be found at the following web address: http://www.sabiosciences.com/manuals/shRNAwhitepaper.pdf The conclusion of this study was that the three most important criteria to meet, in order to establish a reliable RNAi validation protocol, are as follows: Transfection efficiencies of 80% or higher Standard deviation in the technical replicate raw Ct values from the qPCR analyses should be no greater than 0.2. Carry out the experiment with no less than three biological replicates of each target gene-specific siRNA/shRNA and each negative control siRNA/shRNA.
