Gene expression analysis by real-time PCR: Experimental demonstration of PCR detection limits

Reverse transcription followed by real-time PCR (RT-qPCR) is the gold standard for quantifying gene expression. However, because of PCR detection limits, theorized to be three template copies, the quantification of genes exhibiting great expression variability is challenging. Using genes with high to low expression in rat tissues we experimentally demonstrated this limit and found it to be applicable only for describing reactions in which stochastic events and the Monte Carlo effect are present. We also determined the lower limits of RNA input that should be used to prevent artifactual template quantification and we propose a methodology to assess RT-qPCR detection limits in any qPCR platform. (C) 2012 Elsevier Inc. All rights reserved.