15.10: Real-Time PCR
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is carried out either using fluorescent dyes or sequence-specific probes linked to a fluorophore. The fluorescent dyes used in this case can specifically bind to double-stranded DNA and exhibit fluorescence only when bound to DNA. Fluorescence is measured at the end of each PCR cycle after the synthesis of the complementary strand. In the case of fluorophore-labeled probes, fluorescence is exhibited when the fluorophore is cleaved from the probe during the synthesis of the complementary strand. Fluorescence exhibited by these molecules is then detected by photodetectors that convert the fluorescence signals to a readable format. Real-time RT-PCR requires a specialized PCR machine equipped with a detector to enable real-time quantification, as a traditional PCR machine is not equipped for such analysis.
The output can be analyzed in a number of ways. One way is to count the number of cycles required for the fluorescence to reach detectable levels. The threshold cycle, denoted by Ct, is when the fluorescence is detected over and above the background noise. The greater the quantity of the target in the starting material, the faster the significant increase in fluorescence will appear, resulting in a lower Ct. The Ct value finds application in the downstream quantification or detection of the presence or absence of the target sequence. Additionally, comparing the Ct values of samples of unknown concentration with a series of Ct values obtained from standard concentration can determine the amount of template in an unknown reaction. This is known as absolute quantification and can also be carried out by comparing the fluorescence to a standard curve prepared using known DNA concentrations.