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Digital PCR data. Each gray spot is a positive reaction for a different DNA molecule. Credit: Stephen Bustin.
not constraint, and thus allow for common sense, espe- cially when performing large-scale screens of multiple targets. Still, says Bustin, researchers following the qPCR MIQR guidelines need at the very least to test samples for the presence of PCR inhibitors, measure PCR ef fi- ciency, and screen gene panels to identify suitable genes for normalization. “[For] everything else, you just record what you’re doing, but these three things require signifi- cant additional ef fort,” he says. Similarly, the digital MIQE guidelines require users to measure such values as the number of digital PCR partitions, the average number of DNA molecules per partition, their volume, and the vari- ance in that volume—numbers that help researchers and reviewers better understand the quality and reliability of a reaction.
What’s really needed to broaden MIQE compliance,
Bustin says, is the engagement of journal editors. Yet de- spite the flurry of editorials and peer-reviewed research over the past few years on the problem of data reproduc- ibility, Bustin and his colleagues have found that MIQE adherence actually correlates inversely with journal im- pact
factor (although that may be in part because pa-
pers in higher impact journals tend to combine qPCR and dPCR with other types of data, providing a kind of cross- validation, Bizouarn suggests). Nature Methods and BioTechniques make no mention of MIQE in their author guidelines, but PeerJ and Nucleic Acids Research do. Clinical Chemistry actually requires authors to complete and submit a MIQE checklist along with their manuscript.
Interestingly, says Bustin, one group that has been par-
ticularly supportive is instrumentation vendors. Though there’s no such thing as a “MIQE-certified” instrument or kit, many vendors actively promote the guidelines to their customers. Bio-Rad, for instance, sponsors a mobile app,
Vol. 58 | No. 5 | 2015 221
available for both iOS and Android, that quantifies MIQE compliance in real time. Afif Abdel Nour, an applications scientist at Bio-Rad who co-developed the app during his former position at the Institut Polytechnique LaSalle Beauvais, says it provides “a digital checklist.” Users can save the state of a project and export it for submission to a journal or to share with colleagues—and manuscript reviewers can use the app to ensure articles are up to snuf f. The app has been downloaded some 11,000 times for iOS and 3000 times for Android, according to Abdel Nour.
Such tools may make researchers more aware of good
PCR practices. But Huggett suggests MIQE may also face a perception problem. Some researchers, he says, believe the guidelines are “a dictatorship so to speak, or a police state.” Yet MIQE, he notes, is not a set of rules but sugges- tions. Rather than forcing researchers to pile on extra con- trols, the goal is to help them make the most of what they have in order to make every reaction count. And the only way to do that, he says, is by recognizing the limitations of the method and the data it produces. “That to me is the key behind MIQE. It’s about providing that information and making conclusions that are appropriate.”
References
1. Dijkstra, J.R., van Kempen L.C., Nagtegaal, I.D., and Bustin S.A. 2014. Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results? Mol Oncol. 8:813-818.
2. Bustin, S.A. 2015. The reproducibility of biomedical Sleepers awake! Biomol. Detect. Quantif. 2:35-42
research:
Written by Jeffrey Perkel, Ph.D. BioTechniques 58:217-221 (May 2015) doi: 10.2144/000114283
www.BioTechniques.com
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