Real-Time PCR:

  Real-Time PCR Technology Description All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which increases in direct proportion to the amount of PCR product in a reaction. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection by conventional quantitative PCR methods. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. Allied uses a simple and inexpensive detection method of a dye, SYBR Green I, that emits fluorescent light when intercalated into double-stranded DNA. Because intercalating dyes such as SYBR Green I do not make a distinction between the different dsDNA molecules in a PCR reaction, the formation of non-specific amplicons must be prevented. Therefore, accurate primer design and optimization of the reaction conditions for the primers are required. After the PCR reaction, an additional time-temperature program provides a melting curve to detect the presence of high amounts of non-specific sequences. These non-specific sequences show melting peaks different to the template sequences. Performing a melt curve analysis provides a simple control for non-specific sequences detection providing reliable results with the use of intercalating dyes. Typically, in real-time RT-PCR, a standard curve is generated from a dilution series constructed from a "reference" sample. A serial dilution series of the reference sample is used to generate a standard curve. Real-time PCR is performed on both the experimental samples and reference sample. Relative values for target abundance in each experimental sample are then extrapolated from the standard curve generated from the reference sample. Once a project is initiated, Allied will perform a series of optimization reactions for each primer pair using the BioRad iCycler iQ Real-Time PCR Detection System. These optimizations will be done using the reference sample as a standard curve. Parameters optimized include primer concentration and Tm. In some cases, additional optimization will be needed (i.e. [Mg2+]). Once the reaction conditions have been optimized, the RNA samples of interest will be assayed with each primer set. All reactions and standard curves are performed in triplicate in order to statistically assess variability. Allied is available to discuss the application of real-time PCR to your specific research project. We are able to assist with primer design and to offer suggestions for the isolation of high quality RNA. We have successfully applied the Real Time PCR in the quantitation of telomerase activity. (Linker).