Real-time PCR

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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 [http://pathmicro.med.sc.edu/pcr/LinearPCRReaction.htm diagram] in Hunt tutorial).
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 [http://pathmicro.med.sc.edu/pcr/LinearPCRReaction.htm diagram] in Hunt tutorial).
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== Primer selection ==
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For example with the excellent free Primer3 tool on the web applying the following criteria:
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*pair towards 3' end (often more specific, some cDNAs don't contain)
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*pair separated by an exon-exon boundary (reduces genomic background)
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*amplified region must be no biger than 200 bp; usally 60-150 bp
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*GC content: 50-60%
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*min length: 18, max length 24 (best: 20 nt)
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== Reference mRNAs ==
== Reference mRNAs ==

Revision as of 09:19, 26 March 2007

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).


Contents

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).


Primer selection

For example with the excellent free Primer3 tool on the web applying the following criteria:

  • pair towards 3' end (often more specific, some cDNAs don't contain)
  • pair separated by an exon-exon boundary (reduces genomic background)
  • amplified region must be no biger than 200 bp; usally 60-150 bp
  • GC content: 50-60%
  • min length: 18, max length 24 (best: 20 nt)



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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