Brief guide to RT-qPCR

Abstract

RNA quantification is crucial for understanding gene expression and regulation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a widely used technique for RNA quantification because of its practical and quantitative nature, sensitivity, and specificity. Here, we provide an overview of RT-qPCR, focusing on essential reagents, the importance of primer design, the detailed workflow, and data analysis methods. This guide will be useful for scientists who are unfamiliar with RT-qPCR, highlighting key considerations for successful RNA quantification.

Keywords: RNA quantification; Reverse transcription-quantitative polymerase chain reaction.

Fig. 1

Overview of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) workflow. RT-qPCR consists of reverse transcription, qPCR, and analysis steps. Reverse transcription is a process that converts RNA templates into cDNA using primers, reverse transcriptase, RNA templates, dNTPs, MgCl₂, and ribonuclease (RNase) inhibitors. For the qPCR step, DNA polymerase, dNTPs, DNA template (cDNA), sequence-specific primers, and fluorescent dye/probe are used to amplify cDNA. The analysis strategies for RT-qPCR include absolute quantification or relative quantification. cDNA, complementary DNA; dNTPs, deoxynucleoside triphosphates.