Editing Reverse transcription polymerase chain reaction (section)

===One-step RT-PCR===
One-step RT-PCR subjects mRNA targets (up to 6 kb) to reverse transcription followed by PCR amplification in a single test tube. Using intact, high-quality RNA and a sequence-specific primer will produce the best results.

Once a one-step RT-PCR kit with a mix of reverse transcriptase, Taq DNA polymerase, and a proofreading polymerase is selected and all necessary materials and equipment are obtained a reaction mix is to be prepared. The reaction mix includes dNTPs, primers, template RNA, necessary enzymes, and a buffer solution. The reaction mix is added to a PCR tube for each reaction, followed by template RNA. The PCR tubes are then placed in a thermal cycler to begin cycling. In the first cycle, the synthesis of cDNA occurs. The second cycle is the initial denaturation wherein reverse transcriptase is inactivated. The remaining 40-50 cycles are the amplification, which includes denaturation, annealing, and elongation. When amplification is complete, the RT-PCR products can be analyzed with [[gel electrophoresis]].<ref>{{cite web|title=High Transcript Tools OneStep Kit|url=http://www.biotools.eu/reverse_transcription.html|url-status=dead|archive-url=https://web.archive.org/web/20130520151713/http://www.biotools.eu/reverse_transcription.html|archive-date=20 May 2013|access-date=12 December 2012|publisher=Biotools}}</ref><ref>{{cite book|last=Degen|first=Hans-Joachim|url=http://www.roche-applied-science.com/publications/print_mat/pcr_application_manual_3rd_edition.pdf|title=PCR Applications Manual|author2=Deufel, Annette|author3=Eisel, Doris|author4=Grünewald-Janho, Stefanie|author5=Keesey, Joe|publisher=Roche Diagnostics|year=2006|edition=3|pages=135–137}}</ref>

(PCR Applications Manual and Biotools)