AAT Bioquest

How do I do reverse transcriptase PCR?

Posted February 28, 2022


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

Additional resources

Reverse-transcription PCR (RT-PCR)

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