The PCR Blog: The Polymerase Chain Reaction

Ideally, the analysis software supplied with the platform should be as user-friendly as possible but it is also important to check that the software can fully analyse results of the chosen probe chemistry. Some platforms and analysis software suites are biased towards certain chemistries. Some real-time instruments also have specific primer and probe design software that is either supplied with the hardware or available at extra cost. Such software can help simplify and speed up the assay design process and is optimised for that system and reagents. The LightCyclers also have specific relative quantification software that is designed to determine the exact relative nucleic acid concentration normalized to a calibrator sample. This software speeds up and greatly simplifies relative quantification.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: qPCR, qPCR software, qPCR thermal cycler, qRT-PCR, RT-PCR software, software

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