See also:

• Polymerase Chain Reaction ( PCR)
  

In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique 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 messenger RNA (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.

Background

Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through protein synthesis. 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 RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. 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.

Methodology

Typical output of Real-time PCR experiment

Real-time polymerase chain reaction evolved from the polymerase chain reaction technique which was developed to allow the amplification of small numbers of DNA molecules. Real-time PCR involves first isolating mRNA from a particular cell sample before producing a DNA copy of complementary DNA of each mRNA molecule using the enzyme reverse transcriptase. The gene of interest is then further amplified from the cDNA mixture together with a "housekeeping" gene. Housekeeping genes are those whose expression levels remain roughly constant in all cells and tissues and include such genes as actin and Hypoxanthine-guanine phosphoribosyltransferase. The basic idea behind real-time polymerase chain reaction is that the more abundant a particular cDNA (and thus mRNA) is in a sample, the earlier it will be detected during repeated cycles of amplification. Various systems exist which allow the amplification of DNA to be followed and they often involve the use of a fluorescent dye which is incorporated into newly synthesised DNA molecules during Real-time amplification. Real-time polymerase chain reaction machines, which control the thermocycling process, can then detect the abundance of fluorescent DNA and thus the amplification progress of a given sample. Typically, amplification of a given cDNA over time follows a curve, with an initial flat-phase, followed by an exponential phase. Finally, as the experiment reagents are used up, DNA synthesis slows and the exponential curve flattens into a plateau.

As an example of this technique, suppose an individual wishes to measure the expression level of "Gene-A" in two cell samples. After Real-time PCR amplification he or she finds that in sample 1, Gene-A reaches a pre-determined threshold of detection after 18 cycles, known as the "Ct value", whereas in sample 2 it does not reach the threshold until 22 cycles. If the housekeeping gene has a Ct value of 17 in both cases then the difference between Ct values, or ΔCt, will be 1 for sample 1 and 5 for sample 2. In this case Gene-A is more highly expressed in sample 1 than in sample 2.

The above example is quite simplistic and it is rare that a housekeeping gene will have the same Ct value over all samples analysed. A number of software programs and spreadsheets exist which will allow the user to input Ct values and produce a numerical output showing gene expression levels compared between different cell samples, expressed as a fold difference between samples. Such programs also allow statistical analysis of data, such as calculation of standard error and standard deviation.

Applications of real-time polymerase chain reaction

The technology is being applied to rapidly detect the presence of a wide range of infectious diseases, cancer, defective genes, etc in a variety of fields including human diagnosis and agricultural infestations.

The technology may be used in determining how the genetic expression of a particular gene changes over time, such as during germination, in a tissue in response to an administered pharmacological agent, or in response to changes in environmental conditions.

References

• Molecular Biology of the Cell, Alberts, B. et. al, Garland Science
• A-Z of Quantitative PCR, Bustin, S.A. (ed.), IUL Biotechnology, 2004

Further reading

• Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. (1992). "Simultaneous amplification and detection of specific DNA sequences." Biotechnology 10:413–417.
• Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). "Kinetic PCR: Real time monitoring of DNA amplification reactions." Biotechnology 11:1026–1030.
• Real Time PCR Tutorial by Dr Margaret Hunt, University of Southern California, September 5, 2006