Freitag, 11. Oktober 2013

Getting over qPCR's technical hurdles

by Sarah Webb, Ph.D.
BioTechniques, Vol. 55, No. 4, October 2013, pp. 165–168
Even though it has become a “workhorse” technique for quantifying nucleic acids, qPCR continues to be plagued by problems of reproducibility and reliability. Yet when carefully designed, optimized, and validated, qPCR experiments are incredibly accurate, according to Stephen Bustin of Anglia Ruskin University in Chelmsford, UK. However, in far too many cases, researchers don't carefully optimize and validate their assays or report enough information on reagents, primers and procedures for the research community to evaluate their methods.
When Bustin started using qPCR for gene expression profiling in cancer metastases during the late 1990s, he quickly discovered the pitfalls and problems associated with qPCR data— going so far as to write an early review article bringing the issues out in the open. As a result, he was asked to serve as an expert witness in a high profile UK court case where his analysis of qPCR data showed flaws that wrongly linked the MMR vaccine with autism.
Years of discussions focusing on these technical challenges amongst Bustin and like-minded colleagues eventually culminated in a 2009 article published in the journal Clinical Chemistry that provides a set of guidelines for performing qPCR experiments. These guidelines are known as the minimum information for publication of quantitative real-time PCRexperiments (MIQE). Four years later though, only 11%of papers that report qPCR experiments cite those guidelines.

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