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

Cold Spring Harb Protoc. 2018 Oct 1;2018(10). doi: 10.1101/pdb.prot095042.

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.

MeSH terms

  • RNA / analysis*
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcription / genetics

Substances

  • RNA