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It can be daunting to learn about a new topic when you look at the literature and realize that thousands of papers have been published on the topic. This is especially true for techniques that have transformed scientific research.
Quantitative PCR (qPCR) also known as Real-Time PCR (RT-PCR) has revolutionized the study of gene expression by allowing the ‘real-time’ detection and quantitation of a gene(s) of interest.
The breadth of literature available on qPCR is astounding and finding a good starting point can be difficult. So what you really need to get started is a list of the most important/influential papers on the topic. But never fear! To help you out, I have compiled a list of 8 of the most essential papers and reference guides about qPCR. Here goes:
This is a great starting point if you have never performed qPCR before or are training a junior colleague in the technique. It gives a good overview of the principles involved, how you go about designing assays and once they have been performed, how to carry out the data analysis. Qiagen and Bio-Rad both have similar guides on their websites. It can be particularly helpful for a newbie to use the reference guide produced by the company whose reagents you are working with.
This widely cited article from Nature protocols is aimed at scientists whom are performing qPCR on a regular basis and are looking for more specifics. The authors detail protocols that highlight the necessary technical steps required to produce accurate, reliable and reproducible data. They also flag issues that can impede this and offer strong troubleshooting advice.
3. The implications of using an inappropriate reference gene for real time reverse transcription PCR data normalization
4. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
These papers address the use of reference genes such as GAPDH and beta-actin and how their expression can actually vary in response to experimental conditions. A number of examples are given in the first paper and both papers discuss the use of multiple reference genes for more accurate normalisation of data.
Articles 5 and 6 are application notes from Life technologies. The first describes what Ct is, the factors that can influence it and how to evaluate the performance of a real-time PCR reaction by assessing efficiency, precision & sensitivity. The second focuses on PCR efficiency and the importance of following strict protocols.
7. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Articles 7 and 8 outline the difference between relative and absolute quantification of RT-PCR data and describe mathematical models to analyse your data using relative quantification.
These articles and application notes should give you a good start on understanding the theory behind qPCR and hints and protocols for designing and analysing your own experiments.
Are there any articles you swear by for qPCR theory and/or assay development?