A pending subject: normalization of RT-qPCR

Abstract

In recent years, the molecular biology has gone through a series of technical revolutions. The RT-qPCR is a technique with a leading role in one of these revolutions. Since its emergence, the gene expression analyses have become quantitative, cheaper and faster, thus more popular in the researchers’ community spreading its use across all the laboratories. However, the technique is not as easy as the most of researchers think. The incorrect implementation of the RT-qPCR leads to inconsistent and unrepeatable results. Nevertheless, even at this point, there are researchers making efforts to redirect this situation stablishing a series of guidelines as in the case of MIQE standards. One of the most problematic issues is the estimation of the gene expression normalizing the raw results from a RT-qPCR. Usually, almost every researcher consider that the normalization must be performed using reference genes with stable expression in every condition or tissue, so-called “housekeeping” genes. However, this concept has been overcome by the experience showing that the selection of the normalization genes must be achieved through experimental procedures following a “code of good conduct” such as the MIQE guidelines.