Quantitative PCR

Quantitative PCR (also qPCR, or real time PCR, RT-PCR not to be confused with reverse transcription PCR) is a method used for the quantitative detection of DNA or RNA in a sample. In the case of RNA, it is generally referred to as qRT-PCR, meaning quantitative reverse transcription PCR.

qPCR measures the amount of DNA or RNA that has been amplified in real-time by monitoring the intensity of fluorescence emitted (fluorescence intensity should be directly proportional to the amount of PCR product formed). The initial amount of template can be quantified from this data, providing it is taken from a linear phase of the PCR reaction.

qPCR involves the production of small PCR products (in the order of 100bp). This reduces the extension phase of PCR, in order to provide a high turnover for quick detection.

The fluorescence in qPCR can be achieved in a number of ways:

  • Intercalating dyes - e.g. SYBRgreen. These form complexes with duplex DNA and emit more fluorescence compared with the free dye. SYBRgreen is now used preferentially to the traditional ethidium bromide, due to the carcinogenic properties of the latter compound. As more duplex DNA is formed (as would be the case during PCR), more fluorescence is emitted as a result of SYBRgreen binding. However, one problem with SYBRgreen is that it binds to duplex DNA non-specifically and as such, a wrongly-amplified double-stranded DNA (such as a primer dimer) can give a false fluorescence signal.

  • Hybridisation probes, used in conjunction with PCR primers, offer sequence specificity not provided by intercalating dyes. These probes typically have reporter groups, which emit fluorescence, and quencher groups, which suppress the reporter group until activation by a particular mechanism. Taqman is an example of such a probe: it has a 5' reporter and a 3' quencher. When Taq polymerase is added to the probe-template mixture, its 5'-3' exonuclease activity digests Taqman, liberating the 5' reporter which can then emit fluorescence (a confirmation that extension has occurred). Beacons are another example probe. They change their 3D structure upon hybridisation to the target, liberating the reporter group from the quencher group, resulting in fluorescence. FRET (fluorescence resonance energy transfer) involves the use of two probes, one labelled at the 5' end with a red fluorophore and one labelled at the 3' end with fluorescin. When both probes hybridise with the PCR product, they become in close enough proximity for energy donation from fluorescin to the fluorophore (again resulting in fluorescence).

The qPCR machinery involves a tungsten lamp to excite probes in the PCR reaction mixtures, which are contained in a 96-well microtitre plate. The fluorescence emitted is directed through a series of mirrors to a rotating filter, allowing multiple signals to be detected simultaneously.

A threshold can be set for the amount of DNA required from the qPCR reaction. The number of amplification cycles required to obtain the desired amount of DNA is called the cycles for threshold or Ct. Clearly, the lower the starting concentration of template (i.e. the more diluted the initial reaction mixture), the higher the Ct.

A melting plot can be used to ascertain whether only one PCR product has been made (i.e. a measure of the specificity of the PCR primers to the desired template). This involves re-naturing the products at various temperatures, and then measuring the absorbance of the re-natured products by spectrophotometry. Where the absorbance significantly differs from a norm indicates that a contaminating PCR product has been synthesised.

The applications of qPCR are diverse: qRT-PCR, in particular, is clinically useful in the analysis of gene expression and the quantitiation of foreign (e.g. viral) DNA or RNA in a patient's cells.

The use of qRT-PCR in measuring gene expression

qRT-PCR is useful when measuring gene expresion from small amounts of initial sample. Existing methods for transcript quantification (northern blotting, RNase analysis, in-situ hybridisation and RT-PCR) are only semi-quantitative in this respect. Cell RNA is extracted, mRNA is isolated and converted to cDNA, and then cDNA-specific primers are used in PCR amplification. For comparative purposes, it is imperative to also use standard mRNAs whose copy number is expressed constantly in all cells and independently of external conditions (for instance, ribosomal RNA or an mRNA for a glycolytic enzyme).

It is also important to run a host of control reaction mixtures, such as negative control with no cDNA (to check for contaminants), a positive control (to check that reagents and primers work, especially when checking for the absence of gene expression), a control without reverse transcriptase (to ensure that contaminating DNA is not causing a signal), and a 'loading control' (to ensure that an increased signal is not due to a loading artifact - i.e. more RNA being loaded into a gel. With the loading control, a reference gene which has consistent copy number should be loaded in addition to the gene whose expression is under study. If the quantity of reference gene in the experimental lane is 2-fold that in the control lane, then twice as much RNA has been loaded, and if the experimental gene under these conditions is giving a 10-fold signal then the true increase in gene expression is (10/2) = 5-fold.

Quantitation of qPCR results for measurement of gene expression: ΔΔCt method

This involves determining a value called the 'change in change in Ct' or simply, ΔΔCt. It is worth noting that this method only offers an approximation.

A Ct is set for the overall reaction: i.e. an amount of fluorescence that is required for threshold. If there is more starting template, i.e. as there would be if a gene were highly expressed, then the Ct should be lower (as threshold fluorescence is reached in fewer cycles of amplification).

There are two conditions: the control condition and the experimental condition. In both conditions a 'reference' (housekeeping) gene is used for comparative purposes. The difference in Ct (ΔCt) between the reference gene and the target gene is measured in both conditions, and then the difference between the ΔCt values in each condition is measured (ΔΔCt). Assuming efficiency of the reaction is 100%, the ΔΔCt value can then be raised to the power 2 (i.e. 2 ΔΔCt) to give the fold-change in the the target gene (e.g. a ΔΔCt of 11.40 at 100% efficiency gives a fold-change in gene expression of 2^ΔΔCt = 2^11.40 = 2702-fold).

Efficiency refers to the amplification efficiency of both the target and reference genes. It is established by diluting the cDNA templates and using the dilutions in multiple rounds of qPCR to give a plot of cDNA dilution against ΔCt. If the plot is close to zero then the efficiency of the genes is very similar, and it is more acceptable to use the 2ΔΔCt method described above. If a housekeeping gene with similar efficiency cannot be found, then it is preferable to use the standard curve method described below.

Quantitation of qPCR results for measurement of gene expression: Standard curve method

Quite simply, a standard curve of absorbance is constructed from RNA (or preferably cDNA) of known concentration. The curve is then used as a reference standard for extrapolating quantitative information of mRNA (or cDNA) targets of unknown concentration. This method is generally more accurate than ΔΔCt.