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

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.

• Publication year

  2018

• Venue

  Cold Spring Harbor Protocols

• Publication date

  2018-10-01

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1101/pdb.prot095042PMID 30275077
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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