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Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR), a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification". In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR/qRT-PCR), which is also sometimes (incorrectly) abbreviated as RT-PCR.

Contents

RT-PCR principles and procedure

RT-PCR utilizes a pair of primers, which are complementary to a defined sequence on each of the two strands of the cDNA. These primers are then extended by a DNA polymerase and a copy of the strand is made after each cycle, leading to logarithmic amplification [1].

RT-PCR includes three major steps. The first step is reverse transcription (RT), in which RNA is reverse transcribed to cDNA using reverse transcriptase and primers. This step is very important in order to perform PCR since DNA polymerase can act only on DNA templates [1]. The RT step can be performed either in the same tube with PCR (one-step PCR) or in a separate one (two-step PCR) using a temperature between 40°C and 50°C, depending on the properties of the reverse transcriptase used [2].

The next step involves the denaturation of the dsDNA at 95°C, so that the two strands separate and the primers can bind again at lower temperatures and begin a new chain reaction. Then, the temperature is decreased until it reaches the annealing temperature which can vary depending on the set of primers used, their concentration, the probe and its concentration (if used), and the cations concentration. The main consideration, of course, when choosing the optimal annealing temperature is the melting temperature (Tm) of the primers and probes (if used). The annealing temperature chosen for a PCR depends directly on length and composition of the primers. This is the result of the difference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). An annealing temperature about 5 degrees below the lowest Tm of the pair of primers is usually used [3].

The final step of PCR amplification is DNA extension from the primers. This is done with thermostable Taq DNA polymerase, usually at 72°C, the temperature at which the enzyme works optimally. The length of the incubation at each temperature, the temperature alterations, and the number of cycles are controlled by a programmable thermal cycler. The analysis of the PCR products depends on the type of PCR applied. If a conventional PCR is used, the PCR product is detected using agarose gel electrophoresis and ethidium bromide (or other nucleic acid staining).

Conventional RT-PCR is a time-consuming technique with important limitations when compared to real-time PCR techniques [4]. This, combined with the fact that ethidium bromide has low sensitivity, yields results that are not always reliable. Moreover, there is an increased cross-contamination risk of the samples since detection of the PCR product requires the post-amplification processing of the samples. Furthermore, the specificity of the assay is mainly determined by the primers, which can give false-positive results. However, the most important issue concerning conventional RT-PCR is the fact that it is a semi- or even a low-quantitative technique, whereas the amplicon can be visualized only after the amplification ends.

Real-time RT-PCR provides a method in which the amplicons can be visualized as the amplification progresses using a fluorescent reporter molecule. There are three major kinds of fluorescent reporters used in real time RT-PCR, which are general non-specific DNA Binding Dyes such as SYBR Green I, TaqMan Probes and Molecular Beacons (including Scorpions).

The real-time PCR thermal cycler has a fluorescence detection threshold, below which it cannot discriminate the difference between an amplification generated signal and background noise. On the other hand, the fluorescence increases as the amplification progresses and the instrument performs data acquisition during the annealing step of each cycle. The number of amplicons will reach the detection baseline after a specific cycle, which depends on the initial concentration of the target DNA sequence. The cycle at which the instrument can discriminate the amplification generated fluorescence from the background noise is called the threshold cycle (Ct). The higher the initial DNA concentration, the lower its Ct will be.

Use of reverse transcription polymerase chain reaction

The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot analysis is used to study the RNA's gene expression further. RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Due to the fact that most eukaryotic genes contain introns which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error prone nature of reverse transcriptases) DNA sequence which would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons (prokaryotes, such as E.coli, lack the mRNA splicing mechanism of eukaryotes).

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV.

See also

References

  1. ^ a b Hunt M. (2006) Real time PCR tutorial - Copyright 2006, The Board of Trustees of the University of South Carolina
  2. ^ Bustin SA (2000). "Journal of Molecular Endocrinology, 25". Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. pp. 169–193. 
  3. ^ Innis MA et al. (1990). "Academic Press". PCR Protocols: A Guide to Methods and Applications. 
  4. ^ Mackay IM, Arden EK and Nitsche A (2002). "Nucleic Acids Research, vol.30 no.6". Real-time PCR in virology.. pp. 1292–1305. 

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