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 benefi cial in the long run. Specifi city, sensitivity, effi ciency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, ...

Real-time PCR is a powerful tool to compare relative transcriptional abundance, and has been broadly applied in biomedical sciences. However, methods for rigorous statistical analyses have lagged behind applications. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other usefu...

The real-time polymerase chain reaction (real-time PCR) is a recent modification to PCR (UNIT 10.2) that is rapidly changing the nature of how biomedical science research is conducted. It was first introduced in 1992 by Higuchi and coworkers and has seen a rapid increase in its use since (Higuchi et al., 1992, 1993). Real-time PCR allows precise quantification of specific nucleic acids in a com...

Unlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the s...

We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over conta...