Editing Reverse transcription polymerase chain reaction (section)

==== 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>