Editing Reverse transcription polymerase chain reaction (section)

=== 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}}