Reverse transcriptase PCR (RT-PCR) consists of 5 basic steps:
Step 1- Sample preparation
For two-step RT-PCR, the first step is to extract RNA. It’s best to use a ready-to-use RNA extraction kit for this purpose to avoid contact with RNase during extraction. You get higher yield and improved performance with a ready-to-use RNA extraction kit.
Step 2- Selection of primers
Choosing the correct primer for your assay is crucial for best results. There are three types of primers you can choose from:
- Random primers – Random primers are effective for amplification of rRNA, tRNA, prokaryotic RNA and other smaller RNAs but are less effective for eukaryotic RNA amplification as they are unable to bind to a poly-A tail.
- Oligo(dT) primers – These are especially designed for amplification of mRNA, which has a poly-A tail. The oligo(dT) primers are capable of amplifying the entire mDNA into cDNA. They can also be used to amplify smaller mRNAs.
- Sequence-specific primers – Sequence-specific primers can only amplify a specific region and are generally used to amplify a gene of interest in one-step RT-PCR.
Step 3- Reaction preparation
This step involves careful selection of all components required to carry out the RT-PCR reaction. This includes:
- DNA polymerase
- DNA primers
- Template nucleic acid
- Reverse transcriptase enzyme with RNase activity
- dNTPs
- DNA ligase
- RT-qPCR buffer with PCR enhancers and RNase inhibitors
- DEPC treated nuclease-free water
Step 4- RT-PCR cyclic condition
This involves three main steps. First the primers are annealed at temperatures of 55°C - 65°C for 5 minutes to facilitate binding to the RNA template. The reaction is then placed for cooling at 4°C for proper binding. The second step is polymerization, in which the reaction is maintained at 10°C - 50°C for 10 to 90 minutes. Deactivation, which is the third step, involves maintaining the reaction at 85°C for 10 minutes.
Step 5- Strand synthesis
This is the most crucial step and is performed over two stages. First-strand cDNA synthesis forms the first stage. During this stage the cDNA from the single-stranded RNA is amplified by the reverse transcriptase enzyme. This is followed by second-strand cDNA synthesis, which forms the second stage. During this stage, special enzymes such as DNA polymerase add dNTPs to fill the nicks created by RNase activity. DNA ligase