Real-time polymerase chain reaction

In Molecular Biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on polymerase chain reaction, 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 a specific sequence in a DNA sample.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. 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 organisms regulate their cellular activities by activating or deactivating the expression of their genes. Gene expression levels determine the number of copies of messenger RNA (mRNA) of a particular gene in a cell or tissue. As mRNA becomes translated at the ribosome to produce proteins, mRNA levels tend to be roughly correlated with protein levels.

Traditionally, the amount of a particular mRNA produced, and thus the expression level of a gene has been estimated by a technique known as northern blotting. In this method, purified RNA is separated by agarose gel electrophoresis, and then probed with a specific DNA 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 thus cannot be performed when RNA amounts are limited.

In order to detect gene expression at minute levels from single or small numbers of cells, amplification of the gene transcript is necessary. The polymerase chain reaction is a common method for amplifying DNA. However, for mRNA-based PCR the RNA sample first needs to be reverse transcribed to cDNA via the enzyme, reverse transcriptase.

Development of PCR technology based on reverse transcription that uses fluorophores permits measurement of DNA amplification in real time. In real-time PCR, the amplified product is measured at the end of each PCR cycle. The data can be analysed by computer software to calculate relative gene expression between several samples, or mRNA copy number based on a standard curve.

Real-time PCR using double-stranded DNA dyes
A DNA-binding dye binds to all double-stranded (ds)DNA in a PCR reaction, 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.


 * 1) The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
 * 2) The reaction is run in a thermocycler, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.

Like other real-time PCR methods, the values obtained do not have absolute units associated with it (i.e. mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.

Fluorescent reporter probe method
Using fluorescent reporter probes is the most accurate and most reliable of the methods, but also the most expensive. It uses a sequence-specific RNA or DNA-based probe to quantify only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and allows quantification even in the presence of some non-specific DNA amplification. This potentially allows for multiplexing - assaying for several genes in the same reaction by using specific probes with different-coloured labels, provided that all genes are amplified with similar efficiency.

It is commonly carried out with an RNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.


 * 1) The PCR reaction is prepared as usual (see PCR), and the reporter probe is added.
 * 2) As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
 * 3) Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
 * 4) Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.



Quantitation
Quantitating 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 quantitation. Also, over the 20-40 cycles of a typical PCR reaction, the amount of product reaches a plateau determined more by the amount of primers in the reaction mix than by the input template/sample.

Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. Since the quantity of DNA doubles every cycle during the exponential phase, relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template.

Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by RT-PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA. As mentioned above, to accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples. This normalization permits accurate comparison of expression of the gene of interest between different samples, provided that the expression of the reference (housekeeping) gene used in the normalization is very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues.

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

Diagnostically real-time PCR is applied to rapidly detect the presence of genes involved in infectious diseases, cancer and genetic abnormalities. In the research setting, real-time PCR is mainly used to provide highly sensitive 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.

Also, the technique is used in Environmental microbiology, for example to quantify resistance genes in water samples.