Real-time PCR: Difference between revisions

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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.
{{back to protocols}}
{{dablink|[[QRT-PCR]]}}


== Principle ==
'''Real-time PCR''' is a [[PCR techniques|PCR technique]] used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR ([[Q-PCR]]).


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 [http://pathmicro.med.sc.edu/pcr/LinearPCRReaction.htm 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.
Real-time PCR is often preceded by reverse transcription to quantifiy RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called [[qRT-PCR]] for '''q'''uantitative '''r'''everse '''t'''ranscription PCR.


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.
== 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 [http://pathmicro.med.sc.edu/pcr/LinearPCRReaction.htm 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 quantification of the gene of interest.


== Primer selection ==
Real-time 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).


For example, with the excellent free Primer3 tool on the web ([http://fokker.wi.mit.edu/primer3/input.htm Primer3 v0.3], [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer3Plus]). Apply the following criteria:
Generation of product is detected in one of two ways <cite>MeasuringGeneExpression</cite>.  First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction.  Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.
*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-w: 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.  
==Notes==
*When using [[SYBR Green I]] to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product <cite>MeasuringGeneExpression</cite>.
*When using [[SYBR Green I]], the amount of fluorescence in a PCR product depends on the length and base composition of the product <cite>MeasuringGeneExpression</cite>. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.


===Primer repositories===
== See also ==
[http://medgen.ugent.be/rtprimerdb/ 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.
* [[PCR]], [[PCR techniques]], [[Q-PCR]] and [[qRT-PCR]] on OpenWetWare.
 
* An excellent, detailed [http://pathmicro.med.sc.edu/pcr/realtime-home.htm Q-PCR tutorial] by Margaret and Richard Hunt, University of South Carolina
[http://www.realtimeprimers.org/ RealTimePrimers.org] is another such database but it contains only few primers.
* Wikipedia entry on [[wikipedia:Quantitative polymerase chain reaction|Q-PCR]]
 
* The venerable [http://tech.groups.yahoo.com/group/qpcrlistserver/ qpcrlistserv]. Anyone doing qPCR should be subscribed to this list.
See also: [[Designing primers]]
 
==PCR==
 
See also: [[PCR]]
 
== 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 linked to known mouse primer pairs:
==References==
* &beta;-actin (common cytoskeletal enzyme) [http://medgen.ugent.be/rtprimerdb/search_results.php?id=&application=-1&organism=2&gene=actin&string_type=substring&detection=-1&primer=&locuslink=&snp=&last_name=&pubmed=&order=0&search=Search+Primers&first_result=0], [http://www.realtimeprimers.org/SYBR%20Green/Mouse%20SYBR%20Green.html]
<biblio>
* glyceraldehyde-3-phosphate dehydrogenase GAPDH (common metabolic enzyme) [http://medgen.ugent.be/rtprimerdb/search_results.php?primer=&application=-1&detection=-1&locuslink=&snp=&last_name=&order=0&search=Search+Primers&first_result=0&pubmed=&string_type=substring&gene=gapdh&organism=2&Search=Search&id=], [http://www.realtimeprimers.org/SYBR%20Green/Mouse%20SYBR%20Green.html]
#MeasuringGeneExpression isbn=0415374723
* ribosomal proteins (e.g. RPLP0) and RNAs (28S or 18S)
</biblio>
* cyclophilin mRNA
* MHC I (major histocompatibility complex I)


Search [http://medgen.ugent.be/rtprimerdb/ RTPrimerDB] and check the literature before doing it from scratch.
[[Category:Protocol]]  
 
[[Category:In vitro]]  
== See also ==
[[Category:DNA]]  
 
[[Category:RNA]]
* excellent, detailed [http://pathmicro.med.sc.edu/pcr/realtime-home.htm Q-PCR tutorial] by Margaret and Richard Hunt, Uni of South Carolina
* Wikipedia entry on [[wikipedia:Quantitative polymerase chain reaction|Q-PCR]]
* OWW: [[PCR techniques]]

Latest revision as of 10:30, 23 February 2009

back to protocols
      You may be looking for QRT-PCR.

Real-time PCR is a PCR technique used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR (Q-PCR).

Real-time PCR is often preceded by reverse transcription to quantifiy RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called qRT-PCR for quantitative reverse transcription PCR.

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 quantification of the gene of interest.

Real-time 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 in one of two ways [1]. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye SYBR Green I). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.

Notes

  • When using SYBR Green I to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product [1].
  • When using SYBR Green I, the amount of fluorescence in a PCR product depends on the length and base composition of the product [1]. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.

See also

References

  1. ISBN:0415374723 [MeasuringGeneExpression]
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