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 detection techniques include dsDNA dyes like SYBR Green and DNA probe. The cycling step is often preceded by reverse transcription to detect RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses. The sub-technique sometimes carries the bulky label qRT-PCR or even longer names.

Contents

Principle

Amplification of DNA is exponential in the early and middle cycles of a PCR (i.e. it is linear on a logarithmic scale). This property can be exploited to infer the starting amount of PCR template (see diagram in Hunt tutorial). During the exponential or log phase each copy of DNA is being amplified, and thus can be a better measure than in endpoint PCR, where reagents such a nucleotides may become exhausted and result in inefficient amplification, resulting in inaccurate quantitation of the gene of interest.

Quantitative PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR). Generation of product is detected using fluorescent markers such as the DNA binding dye SYBR Green. The intensity of fluorescence is proportional to the quantity of DNA present in the reaction.

Primer selection

An excellent and fast way to select primers is with the free online-tool Primer3, currently in v0.3. Primer3Plus, a variation of Primer3 has qPCR settings. Or just apply the following or similar settings to Primer3:

  • pair towards 3' end (often more specific, some cDNAs don't contain)
  • pair separated by an exon-exon boundary (reduces genomic background) e.g. last exon & penultimate
  • 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)
  • melting temperature: min 60, max 63, best 60
  • max Tm difference: 10 (shouldn't be more than 1 in final pair)
  • max 3' self complementary: 1
  • max poly-x: 3

Verify by blasting the primers sequences. Target gene should come out with the lowerst E value. No other gene should be close. Also check whether possible isoforms will be detected by the candidate primer pair.

Primer repositories

  • RTPrimerDB is an excellent database storing known primer and probe sequences for popular techniques (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon). This can help you save the time of designing and testing your own primers. It is also intended to facilitate standardisation among different laboratories. The database is hosted by the Center for Medical Genetics, Gent, Belgium. Please submit you tested primer pairs.
  • PrimerBank. From the website "PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). PrimerBank contains over 306,800 primers covering most known human and mouse genes. ...The primer design algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. We have tested 26,855 primer pairs that correspond to 27,681 mouse genes by Real Time PCR followed by agarose gel electrophoresis and sequencing of the PCR products. The design success rate is 82.6% (22,187 successful primer pairs) based on agarose gel electrophoresis". Don't neglect to check the efficiency and specificity of the oligos yourself though. Link to paper.
  • qPrimerDepot. From the website "This database provides qRT PCR primers for 99.96% human RefSeq sequences. For 99% of intron-bearing genes, the PCR product will cross an exon-exon border which overlaps one of the largest introns. All primers have annealing temperatures approximately 60C". Link to paper.

See also: Designing primers

Protocols

Reference mRNAs (see Talk)

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 (or similar copy number to gene of interest)

Common reference mRNAs linked to known mouse primer pairs:

  • β-actin (common cytoskeletal enzyme) [1], [2]
  • glyceraldehyde-3-phosphate dehydrogenase GAPDH (common metabolic enzyme) [3], [4]
  • ribosomal proteins (e.g. RPLP0) and RNAs (28S or 18S)
  • cyclophilin mRNA
  • MHC I (major histocompatibility complex I)
  • Search RTPrimerDB and check the literature before doing it from scratch.

See also

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