## RT-PCR allows determination of the input amount of nucleic acid in the reaction

In connection with the test for COVID-19, there has been a lot of talk in public media about PCR testing or RT-PCR testing. The abbreviation RT stands for real-time, so RT-PCR means real-time PCR reaction. PCR is an exponential reaction, so its progress is expressed by an exponential function (Figure 4.3). The number of products increases slowly at first, followed by a sharp increase. Since the reaction has its limits, after a certain time the individual components, especially free nucleotides, are consumed until finally the number of copies of the product no longer increases (plateau phase). Depending on the reaction setup, this usually occurs between the 30th and 40th PCR cycle. This is usually the phase in which we analyse the resulting product of standard PCR, for example by agarose gel electrophoresis.

In RT-PCR analysis, fluorescent dyes that can incorporate into double-stranded DNA are added to the reaction. In their free state, they do not emit a signal (we cannot detect them), but when they are embedded, their activation occurs, and we can record the signal. The process of RT-PCR is then very similar to classical PCR. At high temperature, the double-stranded DNA is denatured, after lowering the temperature, primers are attached (annealed) and new strands are synthesised with the participation of the enzyme DNA polymerase, while a fluorescent dye is incorporated into the product. These steps are also repeated in 30-40 consecutive cycles. In contrast to standard PCR, in RT-PCR the fluorescence signal is evaluated after each cycle and its intensity is recorded.

It is obvious that for this reason an ordinary thermocycler is not sufficient for RT-PCR, but a special type is needed that can record the fluorescence signal - light of different wavelengths. We then see the PCR reaction as an amplification curve formed by combining the measured fluorescence intensities after each cycle. In RT-PCR analysis, the key is the so-called CT value, i.e., the cycle number at which the fluorescence intensity in the sample exceeds a well-defined value that is higher than the background fluorescence. If we analyse different samples in the instrument simultaneously, each may have a different amplification curve and thus a different CT value (Figure 4.4). Here, the less input DNA present at the beginning, the longer it takes for the fluorescence to exceed the threshold. Thus, by precisely analysing the course of the reaction, the amount of DNA used can be determined with relative accuracy. For this reason, RT-PCR is sometimes also referred to as quantitative PCR (qPCR).