Nucleic Acids Research, 2003, Vol. 31, No. 14 e73
© 2003 Oxford University Press
Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis
Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
*To whom correspondence should be addressed. Tel: +44 208 846 7524; Fax: +44 208 846 7506; Email: r.foster{at}ic.ac.uk
Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.
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