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Reverse Transcription PCR

Not all genes are constantly expressed in all cells. A cell must transcribe the DNA sequence into mRNA in order to produce the encoded protein. Therefore, the level of gene expression corresponds to the level of mRNA. Gene expression can be measured using a reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is comprised of these steps:

  • Conversion of RNA into cDNA using reverse transcriptase

  • Amplification of cDNA using PCR

This image illustrates how to create complementary D N A from a messenger R N A template. First, there is a m R N A chain represented as a line, which is incubated with an oligo primer and the enzyme reverse transcriptase. The step 1 is the incubation aforementioned. The result are two lines bound together by numerous perpendicular smaller lines, like a laying ladder. These two lines represent the m R N A and the generation of c D N A using the first chain as a template. The second step is the hydrolization of m R N A once the c D N A strand is completed. The result is half of the ladder corresponding to the c D N A.

Figure 1. Steps to create cDNA from an mRNA template. Using an Oligo(dT) primer, mRNA is reverse transcribed into cDNA, followed by hydrolyzation of the RNA strand.

mRNA is more fragile than DNA and cannot be amplified by PCR. For that reason, mRNA is converted into complementary DNA (cDNA). cDNA is DNA that is synthesized from messenger RNA molecules. cDNA synthesis is catalyzed by an enzyme called reverse transcriptase, which uses RNA as a template for DNA synthesis. Reverse transcriptase was initially discovered and isolated from a retrovirus. These viruses contain an RNA genome; therefore the viruses need to produce a cDNA copy of their genome to be compatible with the host cell's molecular machinery.

The RT-PCR reaction comprises the following steps:

  • An oligo(T) fragment is used as a primer in order to bind to the 3’ poly(A)tail of each mRNA.

  • Reverse transcriptase uses RNA as a template to synthesize cDNA strands.

  • The resulting RNA-cDNA hybrid is separated by increasing the temperature.

  • A gene-specific primer anneals to its complementary sequence.

  • DNA polymerase produces the complementary DNA strand, starting from the primer binding site.

  • The strands are separated by increasing the temperature and the PCR cycle is repeated.

At the end of the RT-PCR reaction, there will be billions of copies of the gene of interest if the gene was expressed. The amount of DNA copies can be visualized by gel electrophoresis.