In molecular biology, reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process.

Polymerase chain reaction itself is the process used to amplify specific parts of a DNA molecule, via the temperature-mediated enzyme DNA polymerase.

Reverse transcription PCR is not to be confused with real-time polymerase chain reaction which is often marketed as RT-PCR.

Processes

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In the first step of RT-PCR, called the "first strand reaction," complementary DNA is made from a messenger RNA template using dNTPs and an RNA-dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription. The above components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 37°C.

After the reverse transcriptase reaction is complete, and complementary DNA has been generated from the original single-stranded mRNA, standard polymerase chain reaction, termed the "second strand reaction," is initiated.

1. A thermostable DNA polymerase and the upstream and downstream DNA primers are added.
2. The reaction is heated to temperatures above 37°C to facilitate sequence specific binding of DNA primers to the cDNA
3. Further heating allow the thermostable DNA polymerase ('Transcriptase') to make double-stranded DNA from the primer bound cDNA.
4. The reaction is heated to approximately 95°C to separate the two DNA strands
5. The reaction is cooled enabling the primers to bind again and the cycle repeats.

After approximately 30 cycles, millions of copies of the sequence of interest are generated. The original RNA template is degraded by RNase H, leaving pure cDNA (plus spare primers).

This process can be simplified into a single step process by the use of wax beads containing the required enzymes for the second stage of the process which are melted, releasing their contents, on heating for primer annealing in the second strand reaction.

Usage of reverse transcription polymerase chain reaction

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The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique, where very low copy number of RNA molecules can be detected. Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases and, quantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot is used to study the RNA's gene expression further.

External links

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• Academic and commercial real-time qRT-PCR platform
• RT-PCR protocols from Penn state University
• Database of validated PCR primer sets

Molecular biologyLaboratory techniques Polymerase chain reaction

RT-PCR | RT-PCR | RT-PCR | RT-PCR | 逆轉錄PCR