Quantitative real time PCR is an important step for the validation of expression data generated by microarray analysis and other genomics techniques. This has been facilitated by the development of real time PCR instruments that measure the amount of PCR product produced at each step of the reaction or in "real time". Examples of these include the Roche LightCycler and the Perkin Elmer ABI 7700 Sequence Detection System. These machines support a variety of chemistries for template detection including SYBR green dye intercalation as well as hybridization probes, hydrolysis probes and molecular beacons. SYBR green is currently the most popular real time PCR method due to its relative ease and reliability. Primers sets may be designed using standard primer design algorithms without any modification. As with all PCR amplifications, however, the specific reaction conditions for each set must be optimized, particularly primer concentration, annealing temperature and magnesium chloride concentration. However, many primer sets fail to amplify the desired template despite all attempts to optimize the reaction conditions and a new set of primers must be designed and synthesized. Considering the time and cost of designing and optimizing primer sets along with the relatively large number of candidate genes that are identified by microarray studies, the development of a central repository for primer sets, reaction conditions and even the actual oligonucleotides would benefit all investigators involved in genomics and other types of experiments. Therefore, we would like to use this site to serve the needs of researchers who are interested in both contributing to and taking advantage of information about quantitative real time PCR.
Real Time PCR Applications
Quantitative real time PCR has revolutionized our ability to measure nucleic acid concentrations. Prior to the development of real time PCR technology, quantitative measurements required one to set up multiple PCR reactions in order to capture PCR product at a linear phase of amplification. Separation and quantitation of the PCR products was then done by gel electrophoresis or HPLC. These experiments are quite laborious and the number of manipulations required to achieve proper quantitation increased the likelihood of introducing error. Thus, the development of real time PCR instruments that could measure PCR product at each thermocycle enhanced the ease, accuracy and reproducibility of quantitative PCR. A variety of applications have emerged as a result of the discovery of real time PCR. These include 1) validation of gene expression data obtained by microarray analysis, 2) measurement of DNA copy number, 3) detection and quantitation of viral particles and potentially lethal microorganisms, 4) mutation and SNP analysis, and 5) measurement of residual disease in cancer patients.
In general, real time PCR protocols are similar to that of standard PCR reactions. The same issues apply with respect to producing clean template, designing primers and optimizing reaction conditions. The major difference is the incorporation of an intercalating agent such as SYBR green or the use fluorescent primers for the detection of PCR product. A description of the different real time PCR chemistries is provided by clicking the specific method at the top of this page. In a typical reaction, PCR product is produced exponentially. 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.
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.
Template Preparation for Real Time PCR
A critical aspect of performing real time PCR is to begin with a template that is of high purity. This can be challenging when working with some biological samples. Fortunately, a number of commercial products have been developed to facilitate the isolation of nucleic acids in high purity. Removing contaminating phenol and unwanted DNA are steps to consider. For gene expression studies, reverse transcription must be carried out with high purity reagents and in multiple replicates as this step can introduce variability in template replication. Reverse transcription may be done prior to real time PCR or may be incorporated within the amplification program.