Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

The software provided with real-time PCR instruments allows several types of data analysis:

1. normalisation of the raw data
2. measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
3. the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
4. melting curves are transformed to provide plots of –dF/dT against T (F = fluorescence and T= temperature) in which a peak (melting peak) occurs at the equilibrium temperature for each duplex

In general instrument specific software is easy to use and allows rapid and reproducible data analysis. In addition to the bundled software a range of third party utilities is available to improve the flexibility of real-time PCR data analyses.

from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR

Labels: data analysis, PCR instruments, qPCR, qRT-PCR, quantitation, quantitative real time pcr