Why is the qRT-PCR reproducibility so critical when detecting gene expression knock down in an RNAi experiment?
A 70 percent drop in gene expression translates into an expected difference in threshold cycles values of 1.75 between gene-specific and negative control shRNA transfected cells. The standard deviation in the threshold cycle values must be less than 0.33 in order to reliably detect this difference. If the reproducibility is poor, then the expected difference can be buried in the noise of the variation in the threshold cycle values. For guidance in designing and executing effective qRT-PCR experiments, we recommend reviewing the qPCR section of the QIAGEN FAQ collection.