FEATURES
lines, digital MIQE’s qPCR-focused predecessors, have been cited some 3200 times since publication in 2009, although more than 26,000 qPCR publications have ap- peared over the same time period.
Stephen Bustin, Professor of Molecular Medicine
at Anglia Ruskin University in the UK, who was corre- sponding author on the original MIQE guidlines and se- nior author on the digital MIQE paper, has spent years lecturing about and documenting the problems of qPCR and dPCR transparency and reproducibility. In 2014, he and his colleagues reported that of 179 papers published between January 2006 and August 2013 using qPCR in colorectal cancer biomarker studies, only 8% used more than a single reference gene (as recommended in the MIQE guidelines). Only 13% of the studies indicated if their reference gene choices were validated, and 70% used 1 of only 3 reference genes (ACTB, GAPDH, or 18S RNA), suggesting they were never validated (1).
Adoption issues
In an editorial in the journal Biomolecular Detection and Quantification, a publication Bustin co-founded spe- cifically
he reported that of 10 articles “selected at random” with RT-qPCR data published by journals in the Nature Publishing Group in 2014, none reported such key MIQE details as RNA integrity, RNA purity, reverse transcrip- tase conditions, or PCR efficiency, and all used just a single unvalidated reference gene for transcript normal- ization, despite evidence suggesting the inaccuracy of that approach (2).
to promote high-quality quantitative studies,
Stephen Bustin co-founded a journal specifically to promote high- quality quantitative studies. Credit: S. Bustin.
“It’s a litany,” Bustin says. “Wherever you look, there’s
a problem.” And according to Bustin, the problem is real. Using
the MIQE guidelines, per se, might not be the key to solving every issue of scientific reproducibility, but
for
qPCR and dPCR, the steps these articles lay out can help researchers ensure their data are well document- ed and rigorous, and thus more likely to be accurate. Conversely, ignoring the guidelines can yield data of lower reliability and robustness, thus wasting researcher time and resources—which is especially galling in light of contracting research budgets. “The conclusion from other people,” he says, citing figures published in The Lancet (2), “is that approximately 85% of research fund- ing is wasted.”
Why would researchers choose to publish poor-
quality data? To some extent, many may not know bet- ter. Andrzej Pietrzykowski, Assistant Professor in the Department of Animal Sciences at Rutgers University and a Visiting Scientist at the Martinos Center for Biomedical Imaging at Massachusetts General Hospital, who follows the dPCR MIQE guidelines in his own work, says many researchers are unaware of MIQE, and those who are aware say that following the guidelines set forth in MIQE requires extra work, and thus, more money. Francisco Bizouarn, Global Digital Applications Specialist at Bio- Rad Laboratories, also suspects that researchers may find the MIQE and digital MIQE checklists, with 80-plus items each, “intimidating.”
Finding common ground
Andrzej Pietrzykowski says that many researchers might be unaware of the MIQE guidelines. Credit: Sonia M. Pietrzykowska.
Vol. 58 | No. 5 | 2015 219
Of course, not every reaction requires that every box be checked. The MIQE guidelines strive for transparency,
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