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Reverse transcription polymerase chain reaction
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Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing, as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA. Conversely, qPCR may be used without RT-PCR, for example, to quantify the copy number of a specific piece of DNA.
The combined RT-PCR and qPCR technique has been described as quantitative RT-PCR or real-time RT-PCR (sometimes even called quantitative real-time RT-PCR), has been variously abbreviated as qRT-PCR, RT-qPCR, RRT-PCR, and rRT-PCR. In order to avoid confusion, the following abbreviations will be used consistently throughout this article:
Not all authors, especially earlier ones, use this convention and the reader should be cautious when following links. RT-PCR has been used to indicate both real-time PCR (qPCR) and reverse transcription PCR (RT-PCR).
Since its introduction in 1977, Northern blot has been used extensively for RNA quantification despite its shortcomings: (a) time-consuming technique, (b) requires a large quantity of RNA for detection, and (c) quantitatively inaccurate in the low abundance of RNA content. However, since PCR was invented by Kary Mullis in 1983, RT PCR has since displaced Northern blot as the method of choice for RNA detection and quantification.
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 (>107-fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. 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 and SARS-CoV-2.
In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. The use of RT-PCR for the detection of RNA transcript has revolutionized the study of gene expression in the following important ways:
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. The two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling. On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-step approach. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. It eliminates the steps of pipetting cDNA product, which is labor-intensive and prone to contamination, to PCR reaction. The further use of inhibitor-tolerant thermostable DNA polymerases, polymerase enhancers with an optimized one-step RT-PCR condition, supports the reverse transcription of the RNA from unpurified or crude samples, such as whole blood and serum. 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, the one-step approach is reported to be less accurate compared to the two-step approach. 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. Nevertheless, the one-step approach is a relatively convenient solution for the rapid detection of target RNA directly in biosensing.[citation needed]
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Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing, as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA. Conversely, qPCR may be used without RT-PCR, for example, to quantify the copy number of a specific piece of DNA.
The combined RT-PCR and qPCR technique has been described as quantitative RT-PCR or real-time RT-PCR (sometimes even called quantitative real-time RT-PCR), has been variously abbreviated as qRT-PCR, RT-qPCR, RRT-PCR, and rRT-PCR. In order to avoid confusion, the following abbreviations will be used consistently throughout this article:
Not all authors, especially earlier ones, use this convention and the reader should be cautious when following links. RT-PCR has been used to indicate both real-time PCR (qPCR) and reverse transcription PCR (RT-PCR).
Since its introduction in 1977, Northern blot has been used extensively for RNA quantification despite its shortcomings: (a) time-consuming technique, (b) requires a large quantity of RNA for detection, and (c) quantitatively inaccurate in the low abundance of RNA content. However, since PCR was invented by Kary Mullis in 1983, RT PCR has since displaced Northern blot as the method of choice for RNA detection and quantification.
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 (>107-fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. 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 and SARS-CoV-2.
In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. The use of RT-PCR for the detection of RNA transcript has revolutionized the study of gene expression in the following important ways:
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. The two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling. On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-step approach. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. It eliminates the steps of pipetting cDNA product, which is labor-intensive and prone to contamination, to PCR reaction. The further use of inhibitor-tolerant thermostable DNA polymerases, polymerase enhancers with an optimized one-step RT-PCR condition, supports the reverse transcription of the RNA from unpurified or crude samples, such as whole blood and serum. 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, the one-step approach is reported to be less accurate compared to the two-step approach. 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. Nevertheless, the one-step approach is a relatively convenient solution for the rapid detection of target RNA directly in biosensing.[citation needed]
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