Reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR (reverse transcription-polymerase chain reaction) is a very important test in the field of gene expression and expression diagnostics because it gives researchers a mechanism to test whether any specific gene is turned on (active) or turned off (inactive). RT-PCR is used to locate and quantify known sequences of mRNA in a sample. It is a rapid and sensitive method for analyzing gene expression, for determining the presence or absence of transcripts and for producing cDNA for cloning. In molecular biology, reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. It is the most sensitive technique for mRNA detection and quantitation currently available.
It can also be carried out as one-step RT-PCR in which all reaction components are mixed in one tube prior to starting the reactions. RT-PCR Reverse Transcription PCR (RT-PCR) couples reverse transcription (RT) of RNA with amplification of the resulting cDNA by PCR. RT-PCR is a rapid and sensitive method for analyzing gene expression, for determining the presence or absence of transcripts and for producing cDNA for cloning.
RT-PCR is used to locate and quantify known sequences of mRNA in a sample as it allows the detection and quantification of mRNA. The first step in RT-PCR uses reverse transcriptase and a primer to anneal and extend a desired mRNA sequence. RNA is first reverse transcribed into cDNA using a reverse transcriptase. The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. If the mRNA is present, the reverse transcriptase and primer will anneal to the mRNA sequence and transcribe a complimentary strand of DNA. If a band shows when run on agarose gel for the desired molecular weight, then the mRNA was in fact present in the sample, and the associated gene was being expressed. If a gene is expressed, its mRNA product will be produced, and an associated band will appear in the final agarose gel with the correct molecular weight for the gene.
To do this, RT-PCR is performed with the unknown mRNA alongside standardized samples with known mRNA amounts. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique, where a very low copy number of RNA molecules can be detected. Furthermore, the techniques is widely used in the diagnosis of genetic diseases and semi quantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot is used to study the RNA's gene expression further.
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