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"RT-PCR" redirects here. For real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, see real-time polymerase chain reaction.


Reverse transcription polymerase chain reaction (RT-PCR) is one of many variants of polymerase chain reaction (PCR), in which the technique is commonly used in molecular biology to detect RNA expression levels.[1] RT-PCR is often interchanged with real-time polymerase chain reaction (qPCR) by students and scientists alike.[2] However, they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes. qPCR is also referred to as quantitative PCR,[3] quantitative real-time PCR,[4] and real-time quantitative PCR[5].

Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. The traditional PCR is simply used to make billions of copies of given DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

In addition to qualitative study of gene expression, RT-PCR can be utilized for quantification of RNA, in both relative and absolute terms,[6] by incorporating qPCR into the technique. The combined technique, described as quantitative RT-PCR[7] or real-time RT-PCR[8] (sometimes even quantitative real-time RT-PCR[9]), is often abbreviated as qRT-PCR[10], RT-qPCR[11], or RRT-PCR[12]. Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.[6]


History

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Since its introduction in 1977, Northern blot had been used extensively for RNA quantification despite its shortcomings of: (a) being time-consuming, (b) requiring a large quantity of RNA for detection, and (c) being quantitatively inaccurate in the low abundance of RNA content.[13][14] However, the discovery of reverse transcriptase during the study of viral replication of genetic material led to the development of RT-PCR which has since displaced Northern blot as the method of choice for RNA detection and quantification.[15]

RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing, (b) a wide range (>10^7 fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data.[9] Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus.[16][17]

However, RT-PCR is not without a major flaw of its own. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity[18]. In order to provide accurate detection and quantification of RNA content in a sample, qRT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR.


Principles

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In RT-PCR, the RNA template is first converted into a complementary DNA using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR. RT-PCR is currently the most sensitive method of RNA detection available.[19] The use of RT-PCR for the detection of RNA transcript has revolutionalized the study of gene expression in the following important ways:

  • Made it theoretically possible to detect the transcripts of practically any gene[20]
  • Enabled sample amplification and eliminated the need for abundant starting material that one faces when using northern blot analysis[21][22]
  • Provided tolerance for RNA degradation as long as the RNA spanning the primer is intact[23]

Despite its major advantages, RT-PCR is not without drawbacks. The extreme sensitivity of the technique can be a double edged sword since even the slightest DNA contamination can lead to undesirable results.[24] Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency.[25]


One-Step RT-PCR vs. Two-Step RT-PCR

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The quantification of mRNA using RT-PCR can be achieved as either a one-step or a two-step reaction. The difference between the two approaches lies in the number of tubes used when performing the procedure. In the one-step approach, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube. On the other hand, the two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. However, the starting RNA templates are prone to degradation in the one-step approach, and the use of this approach is not recommended when repeated assays from the same sample is required. Additionally, one-step approach is reported to be less accurate compared to the two-step approach. One major advantage of the two-step approaches is high reproducibility of the analysis demonstrated with correlation coefficients ranging from 0.974 to 0.988. It is also the preferred method of analysis when using DNA binding dyes such as SYBR Green since the elimination of primer-dimers can be achieved through a simple change in the melting temperature. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling.[26]


End-Point RT-PCR vs. Real-Time RT-PCR

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Quantification of RT-PCR products can largely divided into two categories: end-point and real-time.[19] The use of end-point RT-PCR is preferred for measuring gene expression changes in small number of samples, but the real-time RT-PCR has become the gold standard method for validating results obtained from array analyses or gene expression changes on a global scale.[27]


End-point RT-PCR

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The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide,[28][29] P32 labeling of PCR products using phosphorimager,[30] or by scintillation counting.[22] End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative.[31][32]

Relative RT-PCR: Relative quantification of RT-PCR involves the co-amplification of an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, a direct comparisons of relative transcript abundances across multiple samples of mRNA can be made. One precaution to note is that the internal control must be chosen so that it is not affected by the experimental treatment. The expression level should be constant across all samples and with the mRNA of interest for the results to be accurate and meaningful. Because the quantification of the results are analyzed by comparing the linear range of the target and control amplification, it is crucial to take into consideration the starting target molecules concentration and their amplification rate prior to starting the analysis. The results of the analysis are expressed as the ratios of gene signal to internal control signal, which the values can then be used for the comparison between the samples in the estimation of relative target RNA expression.[33][29][32]

Competitive RT-PCR: Competitive RT-PCR technique is used for absolute quantification. It involves the use of a synthetic “competitor” RNA that can be distinguished from the target RNA by a small difference in size or sequence. It is important for the design of the sythetic RNA be identical in sequence but slightly shorter than the target RNA for accurate results. Once designed and synthesized, a known amount of the competitor RNA is added to experimental samples and is co-amplifed with the target using RT-PCR. Then, a concentration curve of the competitor RNA is produced and it is used to compare the RT-PCR signals produced from the endogenous transcripts to determine the amount of target present in the sample.[34][32]

Comparative RT-PCR: Comparative RT-PCR is similar to the competitive RT-PCR in that the target RNA competes for amplification reagents within a single reaction with an internal standard of unrelated sequence. Once the reaction is complete, the results are compared to an external standard curve to determine the target RNA concentration. In comparison to the relative and competitive quantification methods, comparative RT-PCR is considered to be the more convenient method to use since it does not require the investigator to perform a pilot experiment; in relative RT-PCR, the exponential amplification range of the mRNA must be predetermined and in competitive RT-PCR, a synthetic competitor RNA must be synthesized.[35][36][37][25][32]


Real-Time RT-PCR

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The emergence of novel fluorescent DNA labeling techniques in the past few years have enabled the analysis and detection of PCR products in real-time and has consequently led to the widespread adaption of real-time RT-PCR for the analysis of gene expression. Not only is real-time RT-PCR now the method of choice for quantification of gene expression, it is also the preferred method of obtaining results from array analyses and gene expressions on a global scale. Currently, there are four different fluorescent DNA probes available for the real-time RT-PCR detection of PCR products: SYBR Green, TaqMan, Molecular Beacons, and Scorpions. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes, Molecular Beacons and Scorpions generation of fluorescence depend on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moeity to the oligonucleotide substrates.[38]

SYBR Green: When the SYBR Green binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. However, since the dye does not discriminate the double-stranded DNA from the PCR products and those from the primer-dimers, overestimation of the target concentration is a common problem. Where accurate quantification is an absolute necessity, further assay for the validation of results must be performed. Nevertheless, amongst the real-time RT-PCR product detection methods, SYBR Green is the most economical and easiest to use.[27][19]

TaqMan Probes: TaqMan probes are oligonucleotides that have a fluorescent probe attached to the 5' end and a quencher to the 3' end. During PCR amplification, these probes will hybridize to the target sequences located in the amplicon and as polymerase replicates the template with TaqMan bound, it also cleaves the fluorescent probe due to polymerase 5'- nuclease activity. Because the close proximity between the quench molecule and the fluorescent probe normally prevents fluorescence from being detected through FRET, the decoupling results in the increase of intensity of fluorescence proportional to the number of the probe cleavage cycles. Although well-designed TaqMan probes produce accurate real-time RT-PCR results, it is expensive and time-consuming to synthesize when separate probes must be made for each mRNA target analyzed.[39][20][19]

Molecular Beacon Probes: Similar to the TaqMan probes, Molecular Beacons also make use of FRET detection with fluorescent probes attached to the 5' end and a quencher attached to the 3' end of an oligonucleotide substrate. However, whereas the TaqMan fluorescent probes are cleaved during amplification, Molecular Beacon probes remain intact and rebind to a new target during each reaction cycle. When free in solution, the close proximity of the fluorescent probe and the quencher molecule prevents fluorescence through FRET. However, when Molecular Beacon probes hybridize to a target, the fluorescent dye and the quencher are separated resulting in the emittance of light upon excitation. As is with the TaqMan probes, Molecular Beacons are expensive to synthesize and require separate probes for each RNA target.[26]

Scorpion Probes: The Scorpion probes, like Molecular Beacon, will not be fluorescent active in an unhybrdized state, again, due to the fluorescent probe on the 5' end being quenched by the moiety on the 3' end of an oligonucleotide. With Scorpions, however, the 3' end also contains sequence that is complementary to the extension product of the primer on the 5' end. When the Scorpion extension binds to its complement on the amplicon, the Scorpion structure opens, prevents FRET, and enables the fluorescent signal to be measured.[40]

Multiplex Probes: TaqMan probes, Molecular Beacons and Scorpions allow the concurrent measurement of PCR products in a single tube. This is possible because each of the different fluorescent dyes can be associated with a specific emission spectra. Not only does the use of multiplex probes save time and effort without compromising test utility, its application in wide areas of research such as gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and RNA detection, make it an invaluable technique for laboratories of many discipline.[40][41][42]


Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method.[43]

Uses

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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. Because 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.

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Category:Molecular biology Category:Laboratory techniques Category:Polymerase chain reaction

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