New qPCR guide - Good Practice Guide for the Application of Quantitative PCR

Good Practice Guide for the Application of Quantitative PCR (qPCR)

by Nolan T, Huggett J, Sanchez E

The polymerase chain reaction (PCR) is a rapid, sensitive, and rather simple technique to amplify DNA, using oligonucleotide primers, dNTPs and a heat stable Taq polymerase. With the introduction of real-time PCR in the late nineties, the PCR method overcame an important hurdle towards becoming ‘fully quantitative’ (and therefore known as quantitative PCR, or qPCR). Currently, qPCR is regarded as the ‘gold standard’ in the quantitative analysis of nucleic acids, be it DNA, RNA or micro-RNA molecules. The main reasons for its success are its high sensitivity, robustness, good reproducibility, broad dynamic quantification range, and very importantly, affordability.

However, completing qPCR assays to a high standard of analytical quality can be challenging for a number of reasons, which are discussed in detail in this guide. qPCR has a large number of applications in a wide range of areas, including healthcare and food safety. It is therefore of paramount importance that the results obtained are reliable in themselves and comparable across different laboratories.

This guide is aimed at individuals who are starting to use qPCR and realise that, while this method is easy to perform in the laboratory, numerous factors must be considered to ensure that the method will be applied correctly. The guide aims to assist those who are, or will be, using qPCR by discussing the issues that need consideration during experimental design. The guide entails “tried and tested” approaches, and troubleshoots common issues.

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