Real-time PCR

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Quantitative PCR (Q-PCR or qPCR) also known as real-time PCR is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. The techniques include SYBR Green quantitative PCR, probe-based quantitative PCR and quantitative reverse transcriptase PCR (QRT-PCR).


Principle

Amplification of DNA is exponential in the early and middle cycles of a PCR (i.e. it linear on a logarithmic scale). This property can be exploited to infer the starting amount of PCR template (see diagram in Hunt tutorial).

Reference mRNAs

A mRNA used as reference or standard of a Q-PCR (and other experiments) should have the following properties:

  • expressed in all cells
  • constant copy number in all cells
  • medium copy number for more accuracy

Common reference mRNAs:

  • glyceraldehyde-3-phosphate dehydrogenase (common metabolic enzyme)
  • β-actin (common cytoskeletal enzyme)
  • ribosomal proteins (e.g. RPLP0) and RNAs (28S or 18S)
  • cyclophilin mRNA
  • MHC I (major histocompatibility complex I)

See also

  • excellent, detailed Q-PCR tutorial by Margaret and Richard Hunt, Uni of South Carolina
  • Wikipedia entry on Q-PCR
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