Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Abstract

This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 µL (although most manufacturers recommend 50-µL reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. Discussion on preparing high-quality RNA, choosing a priming method, selecting an enzyme, and selecting an endogenous reference gene is also included.