Caister Academic Press

Performing Real-Time PCR

Authors: K. J. Edwards
Abstract: Optimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence. The analysis of the real-time PCR results is also an important consideration and this differs from the analysis of conventional block-based thermal cycling. Real-time PCR provides information on the cycle at which amplification occurs and on some platforms the melting temperature of the amplicon or probe can be determined.
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