Real-Time PCR

Real-Time PCR, also called quantitative (real-time) PCR, is a method of simultaneous DNA quantification and amplification. DNA is specifically amplified by polymerase chain reaction. After each round of amplification, the DNA is quantified. Common methods of quantification include the use of fluorescent dyes that intercalate with double-strand DNA and modified DNA oligonucleotides (called probes) that fluoresce when hybridized with a complementary DNA. Frequently, real-time PCR is combined with reverse transcription-polymerase chain reaction to quantify low abundance messenger RNA, enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type. The combined technique is often called quantitative RT-PCR.

Quantitative Real-Time PCR Assays

Similar to PCR reactions, several steps are required to develop a reliable quantitative PCR assay. These include producing clean template, designing primers and optimizing reaction conditions. For some gene targets, primer sets and optimized conditions have been organized into databases [1] to facilitate assay development. In a typical reaction, PCR product doubles at each cycle of amplification. Because it takes several cycles for enough product to be readily detectable, the plot of fluorescence vs. cycle number exhibits a sigmoidal appearance. At later cycles, the reaction substrates become depleted, PCR product no longer doubles, and the curve begins to flatten. The point on the curve in which the amount of fluorescence begins to increase rapidly, usually a few standard deviations above the baseline, is termed the threshold cycle (Ct value). The plot of Ct versus template is linear, thus a comparison of Ct values between multiple reactions enables one to calculate the concentration of the target nucleic acid. The slope of this line provides a measure of PCR efficiency. Some real time PCR instruments enable users to generate melting curves following the completion of PCR. Melting curves provide an indication of the purity of the reaction product and reveal the presence of primer dimers.

Quantitation

PCR products may be quantitated by generating a standard curve or quantitated relative to a control gene. Real time PCR quantitation based on a standard curve may utilize plasmid DNA or other forms of DNA in which the absolute concentration of each standard is known. One must be sure, however, that the efficiency of PCR is the same for the standards as that of the "unknown" samples. Performing PCR from purified targets can in some cases be more efficient than that observed with complex nucleic acid mixtures. The relative quantitation method is somewhat simpler as it requires the measurement of housekeeper or control genes to normalize expression of the target gene. However, the selection of appropriate control genes can cause problems as they may not necessarily be equally expressed across all unknown samples. This may be circumvented by normalizing measurements to a set of housekeeping genes in order to avoid this variability problem.

Further reading

• Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413–417.
• Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030.