Reverse transcription-PCR (RT-PCR) is the preferred technique utilized to detect and quanitfy mRNA. RT-PCR may also be used in cloning, constructing a cDNA library, amplifying signal during in situ hybridizations, and synthesizing probes. The first part of the reaction, reverse transciption, synthesizes cDNA from RNA. The second step amplifies the synthesized cDNA to easily detectable levels.

Each reagent serves a specific purpose in the RT-PCR.

• Buffer RT-PCR maintains the correct physiological conditions for both the reverse transcriptase and the Taq DNA polymerase to operate.
• Deoxynucleoside triphosphates are the nucleotide precursors that the Taq DNA polymerase and reverse transcriptase use to generate the cDNA.
• DNA primers are specific to the mRNA sequence and the DNA sequence to be amplified and are needed in order for the correct span of DNA to be amplified.
• Reverse transcriptase converts mRNA into cDNA.
• Taq DNA polymerase is a heat resistant DNA polymerase that synthesizes DNA.
• mRNA provides original template for DNA generation.

Once reagents are added, the RT-PCR procedure can begin.

• During the 37°C incubation step, the reverse transcriptase converts the mRNA template into its corresponding ssDNA. This occurs when the DNA primers and the reverse transcriptase bind the mRNA template and replicate the DNA.
• The temperature is raised to 55°C in order to allow the DNA primers to anneal to the newly synthesized ssDNA.
• The temperature is then raised to 72°C in order to allow the Taq DNA polymerase to extend the ssDNA. Taq DNA polymerase operates optimally at this temperature. This creates a double-stranded cDNA molecule.

Once the cDNA molecule is generated the PCR continues in the following manner.

• Temperature is raised to 94°C for the cDNA to denature.
• Temperature is lowered to 55°C for the primers to anneal to the cDNA.
• Temperature is raised to 72°C for the Taq polymerase to extend the DNA.
• These steps are cycled to obtain the amount of DNA needed.

Examination of the RT-PCR products by electrophoresis is a conformational step to determine if the correct DNA was amplified and to determine that no other DNA was amplified in the procedure.

This Flash animation shows how the method of RT-PCR is performed and how some sample data are produced. It uses sound and mouse-over identification to help students learn more and retain the information.

Legend written by:
Patrick Hume
Colorado State University
Fort Collins, Colorado 80521
hume_patrick@hotmail.com