Editing Reverse transcription polymerase chain reaction (section)

===Two-step RT-PCR===

Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step PCR, use only intact, high-quality RNA for the best results. The primer for two-step PCR does not have to be sequence-specific.

==== Step one ====
First combine template RNA, primer, dNTP mix, and nuclease-free water in a PCR tube. Then, add an RNase inhibitor and reverse transcriptase to the PCR tube. Next, place the PCR tube into a thermal cycler for one cycle wherein annealing, extending, and inactivating of reverse transcriptase occurs. Finally, proceed directly to step two which is PCR, or store product on ice until PCR can be performed.

==== Step two ====
Add master mix which contains buffer, dNTP mix, MgCl<sub>2</sub>, Taq polymerase, and nuclease-free water to each PCR tube. Then add the necessary primer to the tubes. Next, place the PCR tubes in a thermal cycler for 30 cycles of the amplification program. This includes denaturation, annealing, and elongation. The products of RT-PCR can be analyzed with gel electrophoresis.<ref>{{cite web|title=RT-PCR Two-Step Protocol|url=http://ocw.mit.edu/courses/biology/7-16-experimental-molecular-biology-biotechnology-ii-spring-2005/labs/rt_pcr_2step.pdf|publisher=MIT|access-date=12 December 2012}}</ref>