Reverse transcriptase PCR (RT-PCR) consists of 5 main steps:
Step 1. Prepare the sample – This involves extracting the RNA when performing two-step RT-PCR. This step is avoided when performing one-step RT-PCR.
Step 2. Select the primers – The type of primer you use will depend on the purpose of the assay:
- Random primers are the best option for amplification of prokaryotic RNA, rRNA, tRNA, and other smaller RNAs.
- Oligo(dT) primers are used for amplification of mRNA, which has a poly-A tail. They may also be used for amplification of smaller RNAs.
- Sequence-specific primers can only be used to amplify a target gene in one-step RT-PCR as they are capable of amplifying only a specific region.
Step 3. Prepare the reaction – This involves selecting all the necessary components such as:
- Template nucleic acid
- DNA polymerase
- DNA primers
- DNA ligase
- Reverse transcriptase enzyme with RNase activity
- RT-qPCR buffer with PCR enhancers and RNase inhibitors
- dNTPs
- DEPC treated nuclease-free water
Step 4. Perform the RT-PCR procedure – This involves three stages – annealing, polymerization, and deactivation- each of which is carried out at a different temperature. The primers are first annealed at 55°C - 65°C for 5 minutes. This allows them to bind to the RNA template. Placing the reaction at a cooler temperature of 4°C enables proper binding. During the polymerization state, the reaction is maintained at 0°C - 50°C for 10 to 90 minutes. In the deactivation stage, the reaction is maintained at 85°C for 10 minutes.
Step 5. Synthesize new DNA strands – This is performed over two stages. The first stage involves amplifying the first-strand cDNA (complementary DNA) from the single-stranded RNA by the reverse transcriptase enzyme. Second-strand cDNA is synthesized in the second stage. This involves the use of enzyme DNA polymerase, which adds dNTPs to fill the nicks created by the RNase activity. The DNA strand is then ligated by DNA ligase, and new DNA is synthesized.