Editing Reverse transcription polymerase chain reaction (section)

== Principles ==

In RT-PCR, the [[RNA]] template is first converted into a [[complementary DNA]] (cDNA) using a [[reverse transcriptase]] (RT). The cDNA is then used as a template for exponential amplification using PCR. The use of RT-PCR for the detection of RNA transcript has revolutionized the study of gene expression in the following important ways:
* Made it theoretically possible to detect the transcripts of practically any gene<ref name="pmid18645596">{{cite journal  |vauthors=Deepak S, Kottapalli K, Rakwal R, etal |title=Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes |journal=Curr. Genomics |volume=8 |issue=4 |pages=234–51 |date=June 2007 |pmid=18645596 |pmc=2430684 |doi= 10.2174/138920207781386960}}</ref>
* Enabled sample amplification and eliminated the need for abundant starting material required when using northern blot analysis<ref name="doi10.1677/jme.0.0290023">{{cite journal | author = Bustin SA | title = Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems | journal = J. Mol. Endocrinol. | volume = 29 | issue = 1 | pages = 23–39 |date=August 2002 | pmid = 12200227 | doi = 10.1677/jme.0.0290023 | doi-access = free }}</ref><ref name="pmid8862813">{{cite journal |vauthors=Souazé F, Ntodou-Thomé A, Tran CY, Rostène W, Forgez P |title=Quantitative RT-PCR: limits and accuracy |journal=BioTechniques |volume=21 |issue=2 |pages=280–5 |date=August 1996 |pmid=8862813 |doi= 10.2144/96212rr01|doi-access=free }}</ref>
* Provided tolerance for RNA degradation as long as the RNA spanning the primer is intact<ref name="doi10.1677/jme.0.0290023"/>

=== One-step RT-PCR vs two-step RT-PCR ===
[[File:One-step vs two-step RT-PCR.jpg|thumb|450px|One-step vs two-step RT-PCR]]
The quantification of [[mRNA]] using RT-PCR can be achieved as either a one-step or a two-step reaction. The difference between the two approaches lies in the number of tubes used when performing the procedure. The two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling.<ref name="pmid16060372">{{cite journal|vauthors=Wong ML, Medrano JF|date=July 2005|title=Real-time PCR for mRNA quantitation|journal=BioTechniques|volume=39|issue=1|pages=75–85|doi=10.2144/05391rv01|pmid=16060372|doi-access=free}}</ref>  On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-step approach. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. It eliminates the steps of pipetting cDNA product, which is labor-intensive and prone to contamination, to PCR reaction. The further use of inhibitor-tolerant [[thermostable DNA polymerase]]s, polymerase enhancers with an optimized one-step RT-PCR condition, supports the reverse transcription of the RNA from unpurified or crude samples, such as [[whole blood]] and [[serum (blood)|serum]].<ref>{{Cite journal|last1=Li|first1=Lang|last2=He|first2=Jian-an|last3=Wang|first3=Wei|last4=Xia|first4=Yun|last5=Song|first5=Li|last6=Chen|first6=Ze-han|last7=Zuo|first7=Hang-zhi|last8=Tan|first8=Xuan-Ping|last9=Ho|first9=Aaron Ho-Pui|last10=Kong|first10=Siu-Kai|last11=Loo|first11=Jacky Fong-Chuen|date=2019-08-01|title=Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis|url=https://www.ijidonline.com/article/S1201-9712(19)30252-8/abstract|journal=International Journal of Infectious Diseases|language=en|volume=85|pages=167–174|doi=10.1016/j.ijid.2019.06.007|issn=1201-9712|pmid=31202908|doi-access=free}}</ref><ref>{{Cite journal|last1=Bachofen|first1=Claudia|last2=Willoughby|first2=Kim|last3=Zadoks|first3=Ruth|last4=Burr|first4=Paul|last5=Mellor|first5=Dominic|last6=Russell|first6=George C.|date=2013-06-01|title=Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus|journal=Journal of Virological Methods|volume=190|issue=1|pages=1–3|doi=10.1016/j.jviromet.2013.03.015|pmid=23541784|issn=0166-0934}}</ref> However, the starting RNA templates are prone to degradation in the one-step approach, and the use of this approach is not recommended when repeated assays from the same sample is required. Additionally, the one-step approach is reported to be less accurate compared to the two-step approach. It is also the preferred method of analysis when using DNA binding dyes such as [[SYBR Green]] since the elimination of [[primer-dimer]]s can be achieved through a simple change in the [[melting point|melting temperature]]. Nevertheless, the one-step approach is a relatively convenient solution for the rapid detection of target RNA directly in biosensing.{{citation needed|date=April 2020}}

=== End-point RT-PCR vs real-time RT-PCR ===
Quantification of RT-PCR products can largely be divided into two categories: end-point and real-time.<ref name="pmid11017702">{{cite journal |vauthors=Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW |s2cid=258810 |title=Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods |journal=Anal. Biochem. |volume=285 |issue=2 |pages=194–204 |date=October 2000 |pmid=11017702 |doi=10.1006/abio.2000.4753 }}</ref> The use of end-point RT-PCR is preferred for measuring gene expression changes in small number of samples, but the real-time RT-PCR has become the gold standard method for validating quantitative results obtained from array analyses or gene expression changes on a global scale.<ref name="pmid11227069">{{cite journal |vauthors=Rajeevan MS, Vernon SD, Taysavang N, Unger ER |title=Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR |journal=J Mol Diagn |volume=3 |issue=1 |pages=26–31 |date=February 2001 |pmid=11227069 |pmc=1907344 |doi=10.1016/S1525-1578(10)60646-0 }}</ref>

==== End-point RT-PCR ====

The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like [[ethidium bromide]],<ref name="pmid8195381">{{cite journal |vauthors=Stone-Marschat M, Carville A, Skowronek A, Laegreid WW |title=Detection of African horse sickness virus by reverse transcription-PCR |journal=J. Clin. Microbiol. |volume=32 |issue=3 |pages=697–700 |date=March 1994 |pmid=8195381 |pmc=263109 |doi= 10.1128/JCM.32.3.697-700.1994}}</ref><ref name="pmid7787020">{{cite journal |author=Minton AP |title=Confinement as a determinant of macromolecular structure and reactivity. II. Effects of weakly attractive interactions between confined macrosolutes and confining structures |journal=Biophys. J. |volume=68 |issue=4 |pages=1311–22 |date=April 1995 |pmid=7787020 |pmc=1282026 |doi=10.1016/S0006-3495(95)80304-8 |bibcode=1995BpJ....68.1311M }}</ref> [[Phosphorus-32|P32]] labeling of PCR products using [[phosphorimager]],<ref name="pmid22544908">{{cite journal |vauthors=Hsu M, Yu EY, Sprušanský O, McEachern MJ, Lue NF |title=Functional analysis of the single Est1/Ebs1 homologue in Kluyveromyces lactis reveals roles in both telomere maintenance and rapamycin resistance |journal=Eukaryotic Cell |volume=11 |issue=7 |pages=932–42 |date=July 2012 |pmid=22544908 |doi=10.1128/EC.05319-11 |pmc=3416500}}</ref> or by [[scintillation counting]].<ref name="pmid8862813"/> End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative.<ref name="pmid18546601">{{cite journal |vauthors=Schmittgen TD, Livak KJ |title=Analyzing real-time PCR data by the comparative C(T) method |journal=Nat Protoc |volume=3 |issue=6 |pages=1101–8 |year=2008 |pmid=18546601 |doi= 10.1038/nprot.2008.73|s2cid=205464270 }}</ref><ref name=Tang>{{Citation | title = Advanced Techniques in Diagnostic Microbiology | author = Tang, Yi-Wei | isbn = 978-1461439691| date = 2012-09-13 | publisher = Springer }}</ref>

; Relative RT-PCR: Relative quantifications of RT-PCR involves the co-amplification of an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, a direct comparison of relative transcript abundances across multiple samples of mRNA can be made. One precaution to note is that the internal control must be chosen so that it is not affected by the experimental treatment. The expression level should be constant across all samples and with the mRNA of interest for the results to be accurate and meaningful. Because the quantification of the results are analyzed by comparing the linear range of the target and control amplification, it is crucial to take into consideration the starting target molecules concentration and their amplification rate prior to starting the analysis. The results of the analysis are expressed as the ratios of gene signal to internal control signal, which the values can then be used for the comparison between the samples in the estimation of relative target RNA expression.<ref name="pmid7787020"/><ref name="Tang"/><ref name="pmid7522722">{{cite journal |vauthors=Gause WC, Adamovicz J |title=The use of the PCR to quantitate gene expression |journal=Genome Research |volume=3 |issue=6 |pages=S123–35 |date=June 1994 |pmid=7522722 |doi= 10.1101/gr.3.6.s123|doi-access=free }}</ref>
; Competitive RT-PCR: Competitive RT-PCR technique is used for absolute quantification. It involves the use of a synthetic “competitor” RNA that can be distinguished from the target RNA by a small difference in size or sequence. It is important for the design of the synthetic RNA be identical in sequence but slightly shorter than the target RNA for accurate results. Once designed and synthesized, a known amount of the competitor RNA is added to experimental samples and is co-amplified with the target using RT-PCR. Then, a concentration curve of the competitor RNA is produced and it is used to compare the RT-PCR signals produced from the endogenous transcripts to determine the amount of target present in the sample.<ref name="Tang"/><ref name="pmid8922627">{{cite journal |vauthors=Tsai SJ, Wiltbank MC |title=Quantification of mRNA using competitive RT-PCR with standard-curve methodology |journal=BioTechniques |volume=21 |issue=5 |pages=862–6 |date=November 1996 |pmid=8922627 |doi= 10.2144/96215st04|doi-access=free }}</ref>
; Comparative RT-PCR: Comparative RT-PCR is similar to the competitive RT-PCR in that the target RNA competes for amplification reagents within a single reaction with an internal standard of unrelated sequence. Once the reaction is complete, the results are compared to an external standard curve to determine the target RNA concentration. In comparison to the relative and competitive quantification methods, comparative RT-PCR is considered to be the more convenient method to use since it does not require the investigator to perform a pilot experiment; in relative RT-PCR, the exponential amplification range of the mRNA must be predetermined and in competitive RT-PCR, a synthetic competitor RNA must be synthesized.<ref name="Tang"/><ref name="pmid12618301"/><ref name="pmid9888974">{{cite journal |vauthors=Halford WP, Falco VC, Gebhardt BM, Carr DJ |title=The inherent quantitative capacity of the reverse transcription-polymerase chain reaction |journal=Anal. Biochem. |volume=266 |issue=2 |pages=181–91 |date=January 1999 |pmid=9888974 |doi=10.1006/abio.1998.2913 |doi-access=free }}</ref><ref name="pmid20301000">{{cite book |author=King N |title=RT-PCR Protocols |chapter=The use of comparative quantitative RT-PCR to investigate the effect of cysteine incubation on GPx1 expression in freshly isolated cardiomyocytes |volume=630 |pages=215–32 |year=2010 |pmid=20301000 |doi=10.1007/978-1-60761-629-0_14 |series=Methods in Molecular Biology |isbn=978-1-60761-628-3 }}</ref><ref name="pmid12498992">{{cite journal  |vauthors=Chang JT, Chen IH, Liao CT, etal |title=A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients |journal=Clin. Biochem. |volume=35 |issue=8 |pages=591–6 |date=November 2002 |pmid=12498992 |doi= 10.1016/S0009-9120(02)00403-4}}</ref>

==== Real-time RT-PCR ====

The emergence of novel fluorescent DNA labeling techniques in the past few years has enabled the analysis and detection of PCR products in real-time and has consequently led to the widespread adoption of real-time RT-PCR for the analysis of gene expression.<ref name="lww/cmj/2021/09050/rt-rt-pc.5">{{cite journal |last1=Lu |first1=Rou-Jian |last2=Zhao |first2=Li |last3=Huang |first3=Bao-Ying |last4=Ye |first4=Fei |last5=Wang |first5=Wen-Ling |last6=Tan |first6=Wen-Jie |title=Real-time reverse transcription-polymerase chain reaction assay panel for the detection of severe acute respiratory syndrome coronavirus 2 and its variants |journal=Chinese Medical Journal |date=5 September 2021 |volume=134 |issue=17 |pages=2048–2053 |doi=10.1097/CM9.0000000000001687 |pmid=34402479 |pmc=8439998 }}</ref> Not only is real-time RT-PCR now the method of choice for quantification of gene expression, it is also the preferred method of obtaining results from [[Microarray analysis techniques|array analyses]] and gene expressions on a global scale.<ref name="iaea/covid_rt-rt-pcr">{{cite web |last1=Jawerth |first1=Nicole |title=How is the COVID-19 Virus Detected using real time reverse transcription–polymerase chain reaction? |url=https://www.iaea.org/newscenter/news/how-is-the-covid-19-virus-detected-using-real-time-rt-pcr |website=[[International Atomic Energy Agency]] |access-date=16 February 2023 |language=en |date=27 March 2020}}</ref> Currently, there are four different fluorescent DNA [[Hybridization probe|probes]] available for the real-time RT-PCR detection of PCR products: [[SYBR Green]], [[TaqMan]], [[molecular beacon]]s, and [[scorpion probe]]s. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes', molecular beacons' and scorpions' generation of fluorescence depend on [[Förster resonance energy transfer|Förster Resonance Energy Transfer (FRET)]] coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates.<ref>{{Cite book | last1 = Holden | first1 = M. J. | last2 = Wang | first2 = L. | chapter = Quantitative Real-Time PCR: Fluorescent Probe Options and Issues | doi = 10.1007/4243_2008_046 | title = Standardization and Quality Assurance in Fluorescence Measurements II | series = Springer Series on Fluorescence | volume = 6 | pages = 489 | year = 2008 | isbn = 978-3-540-70570-3 | chapter-url = https://zenodo.org/record/1232956 }}</ref>

; [[SYBR Green]]: When the SYBR Green binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. However, since the dye does not discriminate the double-stranded DNA from the PCR products and those from the primer-dimers, overestimation of the target concentration is a common problem. Where accurate quantification is an absolute necessity, further assay for the validation of results must be performed. Nevertheless, among the real-time RT-PCR product detection methods, SYBR Green is the most economical and easiest to use.<ref name="pmid11017702"/><ref name="pmid11227069"/>
[[File:Taqman.png|thumb|Taqman probes]]
; [[TaqMan]] probes: TaqMan probes are oligonucleotides that have a fluorescent probe attached to the 5' end and a quencher to the 3' end. During PCR amplification, these probes will hybridize to the target sequences located in the [[amplicon]] and as polymerase replicates the template with TaqMan bound, it also cleaves the fluorescent probe due to polymerase 5'- nuclease activity. Because the close proximity between the quench molecule and the fluorescent probe normally prevents fluorescence from being detected through FRET, the decoupling results in the increase of intensity of fluorescence proportional to the number of the probe cleavage cycles. Although well-designed TaqMan probes produce accurate real-time RT-PCR results, it is expensive and time-consuming to synthesize when separate probes must be made for each mRNA target analyzed.<ref name="pmid11017702"/><ref name="pmid18645596"/><ref name="pmid15613819">{{cite journal  |vauthors=Yang DK, Kweon CH, Kim BH, etal |title=TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus |journal=J. Vet. Sci. |volume=5 |issue=4 |pages=345–51 |date=December 2004 |pmid=15613819 |doi= 10.4142/jvs.2004.5.4.345|doi-access=free }}</ref> Additionally, these probes are light sensitive and must be carefully frozen as aliquots to prevent degradation.
; [[Molecular beacon|Molecular beacon probes]]: Similar to the TaqMan probes, molecular beacons also make use of FRET detection with fluorescent probes attached to the 5' end and a quencher attached to the 3' end of an oligonucleotide substrate. However, whereas the TaqMan fluorescent probes are cleaved during amplification, molecular beacon probes remain intact and rebind to a new target during each reaction cycle. When free in solution, the close proximity of the fluorescent probe and the quencher molecule prevents fluorescence through FRET. However, when molecular beacon probes hybridize to a target, the fluorescent dye and the quencher are separated resulting in the emittance of light upon excitation. As is with the TaqMan probes, molecular beacons are expensive to synthesize and require separate probes for each RNA target.<ref name="pmid16060372"/>
; Scorpion probes: The scorpion probes, like molecular beacons, will not be fluorescent active in an unhybridized state, again, due to the fluorescent probe on the 5' end being quenched by the moiety on the 3' end of an oligonucleotide. With Scorpions, however, the 3' end also contains sequence that is complementary to the extension product of the primer on the 5' end. When the Scorpion extension binds to its complement on the amplicon, the Scorpion structure opens, prevents FRET, and enables the fluorescent signal to be measured.<ref name="pmid15240248">{{cite journal |vauthors=Sharkey FH, Banat IM, Marchant R |title=Detection and quantification of gene expression in environmental bacteriology |journal=Appl. Environ. Microbiol. |volume=70 |issue=7 |pages=3795–806 |date=July 2004 |pmid=15240248 |pmc=444812 |doi=10.1128/AEM.70.7.3795-3806.2004 |bibcode=2004ApEnM..70.3795S }}</ref>
; Multiplex probes: TaqMan probes, molecular beacons, and scorpions allow the concurrent measurement of PCR products in a single tube. This is possible because each of the different fluorescent dyes can be associated with a specific emission spectra. Not only does the use of multiplex probes save time and effort without compromising test utility, its application in wide areas of research such as gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and RNA detection, make it an invaluable technique for laboratories of many discipline.<ref name="pmid15240248"/><ref name="pmid17489437">{{cite journal |vauthors=Ratcliff RM, Chang G, Kok T, Sloots TP |title=Molecular diagnosis of medical viruses |journal=Curr Issues Mol Biol |volume=9 |issue=2 |pages=87–102 |date=July 2007 |pmid=17489437 }}</ref><ref name="pmid11023957">{{cite journal |vauthors=Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE |title=Multiplex PCR: optimization and application in diagnostic virology |journal=Clin. Microbiol. Rev. |volume=13 |issue=4 |pages=559–70 |date=October 2000 |pmid=11023957 |pmc=88949 |doi= 10.1128/cmr.13.4.559-570.2000}}</ref>

Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method.<ref name="pmid16013967">{{cite journal |author=Bustin SA |s2cid=1833811 |title=Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences |journal=Expert Rev. Mol. Diagn. |volume=5 |issue=4 |pages=493–8 |date=July 2005 |pmid=16013967 |doi=10.1586/14737159.5.4.493 }}</ref>