Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Abstract

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone

“…However, as apparent from Table 1, concentrations of tubulin mRNA measured by quantitative PCR can vary rather widely according to tissue, sex and/or hormonal state (e.g., HYPOX). These findings are in agreement with our [19] as well as other [20,21] earlier reports of significant sex, hormonal and non-hormonal effects on the expression levels of such housekeeping genes as cyclophilin, tyrosine aminotransferase, β-actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase and 18S in rat liver [19]. Accordingly, we normalized real-time PCR determined CYP2C11 mRNA concentrations to total RNA (Table 1) as we [22] and others [23] have reported.…”

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

“…Normalisation to GAPDH was already reported as valid (Wall and Edwards 2002), however in most cases this gene proved to be inappropriate as endogenous control in quantification assays (Schmittgen and Zakrajsek 2000;Goidin et al 2001;Kim et al 2003). A maximum variability of GAPDH of 25 fold has been reported (Dheda et al 2004), and according to Bustin (2002) for most experimental conditions the use of GAPDH is inappropriate and should be discontinued. Our results support this conclusion.…”

“…In many cases genes traditionally chosen for the constancy of their expression have gained this reputation based on assays conducted under a limited number of conditions or with relatively few tissues. Recent data in humans shows considerable variation in expression of certain housekeeping genes in different tissues (Vandesompele et al 2002;Bustin et al 2002). A study of the expression levels of ribosomal RNA 18S, GAPDH, actin and tubulin in rice seedlings subjected to UV-irradiation revealed that only 18S ribosomal RNA was a suitable internal control for real time RT-PCR (Kim et al 2003).…”

Overview of molecular testing in non-small-cell lung cancer: mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors

“…qPCR Several different methods of performing qPCR measurements can be used to detect small amounts of RNA or DNA (Bustin, 2002). Using probes that are labeled with both a fluorophore and fluorescence-quenching molecule, amplicon generation can be detected and quantified in real time during a PCR.…”