Editing Reverse transcription polymerase chain reaction (section)

==== 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>