Real-time polymerase chain reaction: Difference between revisions
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Frequently, real-time PCR is combined with [[reverse transcription]] to quantify [[messenger RNA]] and [[non-coding RNA]] in cells or tissues. |
Frequently, real-time PCR is combined with [[reverse transcription]] to quantify [[messenger RNA]] and [[non-coding RNA]] in cells or tissues. |
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'''qPCR''' is the abbreviation used for real-time PCR.<ref name="VanGuilder et al.">{{cite journal |
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| author=VanGuilder HD, Vrana KE, Freeman WM | year=2008 |
| author=VanGuilder HD, Vrana KE, Freeman WM | year=2008 |
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| title=Twenty-five years of quantitative PCR for gene expression analysis |
| title=Twenty-five years of quantitative PCR for gene expression analysis |
Revision as of 14:17, 18 November 2012
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.
Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and non-coding RNA in cells or tissues.
qPCR is the abbreviation used for real-time PCR.[1] Real-time reverse-transcription PCR is often denoted as: qRT-PCR[2][3][4] The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention.[5]
Background
![](http://upload.wikimedia.org/wikipedia/commons/thumb/0/05/Molecular_Beacons.jpg/300px-Molecular_Beacons.jpg)
Cells in all organisms regulate gene expression by turnover of gene transcripts (messenger RNA, abbreviated to mRNA): the amount that a gene is expressed in a cell is determined by the number of copies of an mRNA transcript of that gene and the rate of degradation of the mRNAs.
Older methods were used to measure mRNA abundance: Differential display, RNAse protection assay and Northern blot. Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe that is complementary to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semiquantitative information of mRNA levels.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse transcribed to cDNA with reverse transcriptase.
Development of PCR technologies based on reverse transcription and fluorophores permits measurement of DNA amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression (or mRNA copy number) in several samples. Real-time PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.
Real-time PCR with double-stranded DNA-binding dyes as reporters
A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can potentially interfere with, or prevent, accurate quantification of the intended target sequence.
- The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
- The reaction is run in a real-time PCR instrument, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.
Like other real-time PCR methods, the values obtained do not have absolute units associated with them (i.e., mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.
Fluorescent reporter probe method
![](http://upload.wikimedia.org/wikipedia/commons/thumb/1/1d/TaqMan_Probes.jpg/750px-TaqMan_Probes.jpg)
Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
- The PCR is prepared as usual (see PCR), and the reporter probe is added.
- As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
- Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
- Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to exponential increase of the product is used to determine the quantification cycle (Cq) in each reaction.
Quantification
Quantifying gene expression by traditional DNA detection methods is unreliable. Detection of mRNA on a Northern blot or PCR products on a gel or Southern blot does not allow precise quantification. For example, over the 20-40 cycles of a typical PCR, the amount of DNA product reaches a plateau that is not directly correlated with the amount of target DNA in the initial PCR.
Real-time PCR can be used to quantify nucleic acids by two common methods: relative quantification and absolute quantification. Relative quantification is based on internal reference genes to determine fold-differences in expression of the target gene. Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards.[6]
A commonly-employed method of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale. A threshold for detection of DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called the quantification cycle, Cq, as suggested by the MIQE guidelines.[7] During the exponential amplification phase, the sequence of the DNA target doubles every cycle. For example, a DNA sample whose Cq precedes that of another sample by 3 cycles contained 23 = 8 times more template. However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in Cq with each dilution. The slope of the linear regression is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a Cq difference of 1. The cycle threshold method makes several assumptions of reaction mechanism and has a reliance on data from low signal-to-noise regions of the amplification profile that can introduce substantial variance during the data analysis.[8]
To quantify gene expression, the Cq for an RNA or DNA from the gene of interest is divided by Cq of RNA/DNA from a housekeeping gene in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔΔCt-method[9] and permits comparison of expression of a gene of interest among different samples. However, for such comparison, expression of the normalizing reference gene needs to be very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues.[10][11]Although cycle threshold analysis is integrated with many commercial software systems, there are more accurate and reliable methods of analyzing amplification profile data that should be considered in cases where reproducibility is a concern.[8]
Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification. Methods such as MAK2[12] have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of the original sample concentration. An extension of this approach includes an accurate model of the entire PCR reaction profile, which allows for the use of high signal-to-noise data and the ability to validate data quality prior to analysis.[8]
Applications of real-time polymerase chain reaction
There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and basic research.
Diagnostic real-time PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases,[13] and is deployed as a tool to detect newly emerging diseases, such as new strains of flu, in diagnostic tests.[14]
Real-time PCR is also used by microbiologists working in the fields of food safety, food spoilage and fermentation and for the microbial risk assessment of water quality (drinking and recreational waters) and in public health protection.[15]
In research settings, real-time PCR is mainly used to provide quantitative measurements of gene transcription. The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administration of a pharmacological agent, progression of cell differentiation, or in response to changes in environmental conditions. It is also used for the determination of zygosity of transgenic animals used in research.
References
- ^ VanGuilder HD, Vrana KE, Freeman WM (2008). "Twenty-five years of quantitative PCR for gene expression analysis". Biotechniques. 44 (5): 619–626. doi:10.2144/000112776. PMID 18474036.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ Udvardi MK, Czechowski T, Scheible WR (2008). "Eleven Golden Rules of Quantitative RT-PCR". Plant Cell. 20 (7): 1736–1737. doi:10.1105/tpc.108.061143. PMC 2518243. PMID 18664613.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ Spackman E, Suarez DL (2008). "Type A influenza virus detection and quantitation by real-time RT-PCR". Methods Mol Biol. 436: 19–26. doi:10.1007/978-1-59745-279-3_4. PMID 18370037.
- ^ Gertsch J, Güttinger M, Sticher O, Heilmann J. (2002). "Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds". Pharm Res. 19 (8): 1236–1243. doi:10.1023/A:1019818814336. PMID 12240952.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ edited by Julie Logan, Kirstin Edwards, and Nick Saunders. (2009). Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
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suggested) (help)CS1 maint: multiple names: authors list (link) - ^ S. Dhanasekaran,T. Mark Doherty, John Kenneth and TB Trials Study Group. (2010 Mar). "Comparison of different standards for real-time PCR-based absolute quantification". Immunol Methods. 354 (1–2): 34–9. doi:10.1016/j.jim.2010.01.004. PMID 20109462.
{{cite journal}}
: Check date values in:|year=
(help)CS1 maint: multiple names: authors list (link) CS1 maint: year (link) - ^ Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, and Carl T. Wittwer. (2009 Apr). "The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments". Clin Chem. 55 (4): :611-22. doi:10.1373/clinchem.2008.112797. PMID 19246619.
{{cite journal}}
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instead. - ^ Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H (2006). "Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula". J Mol Med. 84: 901–10. doi:10.1007/s00109-006-0097-6. PMID 16972087.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ Nailis H, Coenye T, Van Nieuwerburgh F, Deforce D, Nelis HJ (2006). "Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR". BMC Mol Biol. 7: 25. doi:10.1186/1471-2199-7-25. PMC 1557526. PMID 16889665.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link) - ^ Nolan T, Hands RE, Bustin SA (2006). "Quantification of mRNA using real-time RT-PCR". Nat. Protoc. 1 (3): 1559–1582. doi:10.1038/nprot.2006.236. PMID 17406449.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ Boggy G, Woolf PJ (2010). Ravasi, Timothy (ed.). "A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data". PLOS One. 5 (8): e12355. doi:10.1371/journal.pone.0012355. PMC 2930010. PMID 20814578.
{{cite journal}}
: CS1 maint: unflagged free DOI (link) - ^ Sails AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
- ^ FDA Authorizes Emergency Use of Influenza Medicines, Diagnostic Test in Response to Swine Flu Outbreak in Humans. FDA News, April 27, 2009.
- ^ Filion, M (editor) (2012). Quantitative Real-time PCR in Applied Microbiology. Caister Academic Press. ISBN 978-1-908230-01-0.
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Further reading
- Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. (1992). "Simultaneous amplification and detection of specific DNA sequences." Biotechnology 10:413–417.
- Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). "Kinetic PCR: Real time monitoring of DNA amplification reactions." Biotechnology 11:1026–1030.
- Filion, M. (2012). "Quantitative Real-time PCR in Applied Microbiology." Caister Academic Press. ISBN 978-1-908230-01-0
- Wawrik B, Paul JH, Tabita FR (2002) Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68:3771-3779.
- Logan J, Edwards K, Saunders N (editors) (2009). Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
{{cite book}}
:|author=
has generic name (help)CS1 maint: multiple names: authors list (link)
External links
- Real Time PCR Tutorial by Dr Margaret Hunt, University of South Carolina, September 5, 2006
- openwetware
- RefGenes Open Access online tool to identify tissue specific reference genes for RT-qPCR