PCR (3): qRT-PCR

Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qRT-PCR) is used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction.

The procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification; this is the "real-time" aspect of it. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.

Frequently, real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance mRNA, enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type.

Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused with reverse transcription polymerase chain reaction, also known as RT-PCR. Here, the RNA strand is first reverse transcribed into its complementary DNA (cDNA), followed by amplification of the resulting DNA using polymerase chain reaction. complement or

When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an mRNA copy of the gene's DNA sequence. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein.

Traditionally, the amount of a particular mRNA produced, and thus the activation status of a gene has been measured by a technique known as Northern Blotting. Although this technique is still regarded as the gold standard for measuring gene expression by many, it relies on having access to relatively large amounts of material either in the form of RNA, or the cells from which it is to be isolated. Often, researchers must work with very small numbers of cells, or even single cells and real-time polymerase chain reaction was developed as a way of measuring gene expression in minute cell or tissue samples. However, the extreme sensitivity of real-time polymerase chain reaction means that researchers must guard against introducing any contamination into their samples which could dramatically affect the final results. Real-time PCR does however have the added advantage that it is not necessary to measure the concentrations of mRNA or cDNA in a sample before exposing it to real-time polymerase chain reaction. The more abundant a particular cDNA and thus mRNA in a sample, the earlier it will be detected during repeated cycles of amplification.

A typical analysis is shown below. The fluorescence is extrapolated to DNA copy numbers.