How to deal with qPCR thermocycler variability when detecting miRNA

Mary Span
CYCLERtest B.V., The Netherlands

Quantative real-time PCR has become a wide spread applied technique for determining gene expression levels over the past decade, both in fundamental research and clinical diagnostics. qPCR is a very powerful technique, but it’s outcome can be strongly influenced by the experimental set up and the used equipment.

The MIQE guidelines, which were introduced in 2009, are a step forward and are encouraging better experimental practice that allows more reliable and unequivocal interpretation of qPCR results.

In the qPCR protocol section of the MIQE guidelines, it is referred that the complete thermocycling parameters and the manufacturer of the used qPCR instrument should be mentioned.

Over the past years we have measured thousands of PCR and qPCR thermocyclers on a worldwide base and have observed large fluctuations in thermal performance of qPCR thermocyclers, even within the same model and brand. We therefore conclude that the just mentioning the thermocycler manufacturer and thermocycling protocol is not sufficient information to be able to reproduce results published confirm the MIQE guidelines.
The impact of these large thermal fluctuations is, to our experience, strongly underestimated in both diagnostic and research laboratories.

Qualification of a thermocycler as being suitable for a particular PCR, as currently already required by the ISO 17025, ISO 15189 and other medical, veterinary and forensic diagnostics regulations is still lacking in the initial version of the MIQE guidelines and therefore a recommend improvement.

Hereby we present a number of easy applicable guidelines for manufacturer independent thermocycler standardization that do allow full intra- and interlaboratory comparisons and exchange of qPCR data between laboratories. The guidelines are conforming to the ISO 17025 and other norms used by accredited laboratories.

Back to RNAi – microRNA – siRNA applications – miRNA normalisation
Bookmark the permalink.

Comments are closed.