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. 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), is often abbreviated as qRT-PCR, RT-qPCR, or RRT-PCR. In order to avoid confusion, the following abbreviations will be used consistently throughout this article: Since the introduction of Northern blot in 1977, it had 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, 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. 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. In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR. QT-NASBA is currently the most sensitive method of RNA detection available. The use of RT-PCR for the detection of RNA transcript has revolutionalized 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. 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. 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. Quantification of RT-PCR products can largely be divided into two categories: end-point and real-time. 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. The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide, P32 labeling of PCR products using phosphorimager, or by scintillation counting. End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative.