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    Real-Time Polymerase Chain Reaction (RT-PCR)
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    Real-Time Polymerase Chain Reaction (RT-PCR)

    Definition

    Real-time polymerase chain reaction is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule.

    Topics of Interest

    See also: Polymerase Chain Reaction (PCR)

    In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

    The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

    Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues

    Abbreviations used for real-time PCR methods vary widely and include RTQ-PCR, Q-PCR or qPCR. Real-time reverse-transcription PCR is often denoted as qRT-PCR,, RRT-PCR, or RT-rt PCR. The acronym RT-PCR commonly denotes reverse-transcription PCR and not real-time PCR, but not all authors adhere to this convention.

    Cells in all organisms regulate gene expression and turnover of gene transcripts (messenger RNA, abbreviated to mRNA), and the number of copies of an mRNA transcript of a gene in a cell or tissue is determined by the rates of its expression and degradation.

    Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe that is complementary to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semiquantitative information of mRNA levels.

    In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse transcribed to cDNA with reverse transcriptase.

    Development of PCR technologies based on reverse transcription and fluorophores permits measurement of DNA amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression in several samples, or mRNA copy number. Real-time PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.

    Real-time PCR using double-stranded DNA dyes: A DNA-binding dye binds to all double-stranded (ds)DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as "primer dimers"). This can potentially interfere with or prevent accurate quantification of the intended target sequence.

    Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.

    Quantifying gene expression by traditional methods presents several problems. Firstly, detection of mRNA on a Northern blot or PCR products on a gel or Southern blot is time-consuming and does not allow precise quantification. Also, over the 20-40 cycles of a typical PCR, the amount of product reaches a plateau determined more by the amount of primers in the reaction mix than by the input template/sample.

    There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and basic research.

    Diagnostic real-time PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as flu, in diagnostic tests.

    In research settings, real-time PCR is mainly used to provide quantitative measurements of gene transcription. The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administration of a pharmacological agent, progression of cell differentiation, or in response to changes in environmental conditions.

    Source: Wikipedia (All text is available under the terms of the GNU Free Documentation License and Creative Commons Attribution-ShareAlike License.)

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