How to ensure significant results from your qPCR studies

December 20, 2011 at 12:22 pm Leave a comment

So you’ve heard all the hoopla about MIQE and how important it is to follow the guidelines when conducting your real time qPCR experiments, (if you haven’t, you better check this out!), but where’s the proof that following MIQE actually makes a difference? After all, qPCR was around for several years before anyone came up with this MIQE stuff. Right? Well…maybe not! As it turns out, qPCR experiments that don’t follow MIQE guidelines can be very difficult for others to reproduce and can even lead to incorrect conclusions in gene expression studies.

In a recently released case study involving breast cancer patients, researchers found that the MIQE guidelines played a central role in obtaining the expected conclusions with a positive control target. The article was written to show readers in a simple, stepwise process how to design a good qPCR experiment that covers the major components of the MIQE guidelines. While each step of the experimental design was found to impact the final conclusion (sample collection, RNA quality and purity and the use of appropriate primers), the most striking result was the impact of reference gene selection on the results. At one extreme, normalization by the commonly used GAPDH and 18S reference genes gave either no significant results of statistically significant data that was opposite to the expected outcome, while other more stable reference genes, (HPRT1 and TBP), gave statistically significant data that supported the conclusions from previously published results with this target.

The study concludes that the application of the MIQE guidelines to qPCR experiments result in reliable, quantifiable and reproducible data. With a growing list of journals that are now requiring the submission of supplemental data supporting adherence to the MIQE guidelines,the publication of qPCR data will become more challenging if they are ignored.

So don’t miss out on significant data. Use MIQE!

To read the case study click here.

Entry filed under: Bio-Rad Tech Note, PCR Technotes. Tags: , , , , .

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