Quantification of mRNA Expression by RT-qPCR

Abstract

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a ''gold'' standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

Diagnostic Performance of Fas Ligand mRNA Expression for Acute Rejection after Kidney Transplantation: A Systematic Review and Meta-Analysis

“…Quantitative PCR, which was first introduced in 1992 by Higuchi et al [48], has been a common technique in basic and clinical transplantation research [49] and is considered to be the most accurate and reliable technique in validating data obtained by other methods [50]. Reference genes are an internal reaction control that have sequences which differ from the target.…”

Pan-cancer analysis identified the prognosis and antioncogene roles of PRDX3 in kidney renal clear cell carcinoma

“…During each cycle, the fluorescence signal in each reaction tube reached its corresponding threshold value, which was known as Ct. The relative quantitative method was used to compare mRNA expressions as follows: ∆Ct=Ct (target gene) -Ct (reference gene) [21].…”