qPCR basics

Why real-time PCR?
How it works
Good and bad sides of qPCR
Where is qPCR used?
How to learn qPCR


Why real-time PCR? [back to top]

The method of choice for nucleic acid (DNA, RNA) quantification in all areas of molecular biology is real-time PCR or quantitative PCR (qPCR for short). The method got its name because the amplification of DNA with polymerase chain reaction (PCR) is monitored in real time. It is, in contrast to the conventional PCR, quantitative, meaning that it enables us to determine the exact concentration (relative or absolute) of the amplified DNA in the sample. On the other hand, in conventional PCR we can see the result of amplification only after the PCR is completed (end-point detection).

Apart from DNA, RNA can be used as a template as well (e.g. in case of gene expression studies or detection of RNA viruses). In this case the RNA needs to be reverse transcribed into DNA (also termed complementary DNA or cDNA) before it is amplified with real-time PCR. There is a term for this combined method: real-time reverse transcription PCR or qRT-PCR (sometimes RT-qPCR) for short.

How it works [back to top]

PCR is a method where an enzyme (thermostable DNA polymerase) amplifies a short specific part of template DNA (amplicon) in cycles. In every cycle the number of short specific sections of DNA is doubled. More on how conventional PCR works can be found here.

In qPCR, the exactly the same procedure happens but with two major differences: first the amplified DNA is fluorescently labelled (usually with cyanine based fluorescent dyes) and second, the amount of the fluorescence is directly proportional to the amount of amplified DNA, which is monitored during the whole PCR process (along all 30 to 45 cycles). The higher the initial number of DNA molecules in the sample, the faster the fluorescence will increase during the PCR cycles (see Images 1 and 2). This is the basic principle of quantitative approach the real-time PCR offers.


Image 1 shows a graphical represantation of qPCR amplification (the first two cycles) as it is going on in the PCR tube. There are different variants of qPCR (also called chemistries) which have slightly different ways of fluorescence labelling. Image shows two most commonly used. On the left side a 5'-exonuclease variant is shown that uses FRET mechanism (fluorescence resonance energy transfer) where a fluorescence of a reporter fluorofore (R) is transferred to a quencher (Q) and not is not emitted whenever reporter and quencher are in proximity (e.q. linked to the same short oligonucleotide - a probe). When the two are dislocated (when the probe is dissolved away by 5'-exonuclease activity of TaqDNA polymerase during PCR elongation), reporter molecule freely emits the fluorescence which can that be detected. On the right side of the image a qPCR variant that uses an intercalating fluorofore is represented. Special intercalating dyes are used that strongly increase emission of fluorescence whenever they are intercalated into a dsDNA.


Image 2 shows amplification plot showing of five samples (S1 to S5). As the target DNA in each sample is being amplified through cycles the fluoresce increases. Sample S1 contained the highest initial number of target DNA molecules, resulting in the fastest increase of fluorescence. Sample S4 contained the lowest initial number of target DNA molecules while S5 did not contain any target DNA.

There are several ways in which the amplified DNA is fluorescently labelled (also known as “qPCR chemistries”) but we are not going to discuss them in greater details here. They all have one thing in common: to enable production of the fluorescent signal during the PCR reaction that is directly proportional to the starting amount of the DNA.

Good and bad sides of qPCR [back to top]

Advantages to conventional PCR:

•Speed: the amplified DNA is being detected during the PCR reaction so there is no need for a separate detection after as is in the case of conventional PCR (e.g. agarose gel electrophoresis with intercalating fluorescent dyes)
•Throughput: qPCR is considered a high throughput method (processing of large numbers of samples in short time), because it is compatible with robotisation of sample preparation (DNA/RNA isloation and loading onto qPCR plates).
•Sensitivity: qPCR is able to distinguish two fold differences in quantity of target DNA molecule. And it can detect down to a few copies of DNA (sometimes even one).
•Lower amounts of starting material: as low as 1/1000 of the amount required for conventional PCR
•Broad dynamic range of quantification: quantification can be performed over several orders of magnitude (up to 10⁷-fold dynamic range)
•Repeatability: high.

Disadvantages of qPCR:

•Cost of equipment: due to the optical components for sensitive fluorescence detection the qPCR machines are 5 to 10-fold more expensive than conventional PCR thermal cyclers
•Cost of chemicals and consumables: qPCR is a very sensitive method therefore the precise composition and high quality of the reaction mixtures is extremely important. This is the reason why ready-to-use reaction mixtures are usually purchased (master mix). Because of the sensitive detection method (fluorescence) a specific set of plastic-ware is required.
•Inhibition of PCR: due to the complex nature of biological samples, imperfect purification processes during isolation of nucleic acids, etc. PCR reaction is sometimes inhibited by so called inhibitors of PCR reaction (DNA polymerase is an enzyme and as such is susceptible to certain compounds that inhibit its activity – polymerisation of DNA). This can complicate the quantification.
•Sensitivity to mistakes: qPCR is extremely sensitive methods and as such sensitive to mistakes. This means that even the slightest mistakes can have significant influence on the final results. The most variable and critical point is the preparation of the samples (DNA extraction and reverse transcription). That is why several control reactions need to be included along samples when performing qPCR.
•Data analysis: data analysis and interpretation of the results is more complicated.

Where is qPCR used? [back to top]

Due to several powerful advantages qPCR has a wide range of applications. The method has also been around long enough so that the research community proved its reliability and robustness and that manufacturers of qPCR machines developed reliable platforms. The most evident is the use of qPCR in molecular diagnostics, where it is slowly displacing conventional methods. It is used to detect, identify and quantify microorganisms that cause diseases (bacteria, viruses and fungi; see Image 3). With qPCR manual labour is reduced and along that carryover contamination and erroneous results. It is also considered a high-throughput method, meaning that large amounts of samples can be processed in less time (384 or 1536 reactions per plate). qPCR has thus proven to be an excellent method in diagnostic laboratories. It has to be noted, though, that the method detects only the presence of DNA or RNA of a microorganism and does not report its viability. Therefore conventional microbiology techniques are sometimes still required along qPCR.


Image 3 shows a Grapevine fanleaf virus (GFLV) as visualised by transmission electron microscope, one of conventional and labourious detection techniques that is being displaced by RT-qPCR (photo: NIB).

qPCR is also used to detect and quantify genetically modified organisms or to perform genotyping. The latter means that different alleles of the same gene or single nucleotide polymorphisms (SNPs) can be detected which can be used as genetic diagnostic or prognostic markers for certain diseases.

A very important field of use are gene expression studies that help us understand the biological processes in various fields of biology, microbiology, medicine and other life sciences. A very useful, almost blockbuster combination is a genome-wide gene expression screening with DNA-microarrays followed by validation of the results with qPCR. DNA microarrays are a very powerful method on its own but they are less sensitive and still require validation. qPCR is therefore a very important research technique.

How to learn qPCR [back to top]

The best way to learn theoretical and practical big and small tricks, HOW-TOs is to experience them from people who know how to use qPCR and have been doing it for years.

Do it in one of the qPCR Experience workshops.