TechNews GUIDING OUR PCR EXPERIMENTS
The MIQE guidelines for qPCR and dPCR have been around for a while now, but few are taking advantage of this resource. Jeffrey Perkel looks at challenge of standardizing PCR.
In digital PCR, reactions are divided into multiple partitions (in this case, droplets), each of which functions as a single reaction vessel. By counting the number of positive and negative reactions and applying a Poisson correction, researchers can assess the abundance of the target sequence in the starting sample. Credit: Bio-Rad Laboratories.
When Mary Alikian, a clinical scientist and “part-time PhD student” at Imperial College London, began her doctoral
research, she decided to use digital PCR (dPCR). Alikian, who was interested in investigating RNA biomarkers asso- ciated with chronic myelogenous leukemia, quickly realized she had a problem though: no one in the lab had experi- ence with dPCR, so no one could teach her how to do it correctly. “I had no clue what things I should consider and what things I should not.”
If she were using traditional PCR, it might not have mat-
tered so much, as PCR can be fairly forgiving. But dPCR, much like real-time PCR (qPCR), is a quantitative methodol- ogy that is considerably more complicated. The method in- volves dedicated instrumentation, protocols, and variables that are not used in the traditional PCR assay, but the re- sulting data can be used to identify everything from dysreg- ulated genes to chromosomal variation. Improperly trained researchers using dPCR to quantify, say, mRNA abundance, will inevitably obtain a number, but whether that number accurately represents the sample itself is less certain.
Digital guide
Fortunately for Alikian, a group of reproducibility-focused researchers had recently developed a kind of roadmap
Vol. 58 | No. 5 | 2015
for dPCR experiments, called the Minimum Information for Publication of Quantitative Digital PCR Experiments (digital MIQE) guidelines. The concept behind digi- tal MIQE was to guide researchers through the “hows” and “whys” of dPCR, including experimental design and analysis. The guidelines even offer specific recommen- dations on how to report dPCR experiments for maxi- mum transparency. Jim Huggett, Principal Scientist for Nucleic Acid Research at the international life sciences company LGC, who was lead author on the digital MIQE paper, calls the guidelines “a call for information.” Digital MIQE, he explains, “is about including in your publication the information that makes it possible for me to repro- duce your work.”
For her part, Alikian viewed digital MIQE as a set of
best practices for producing high-quality, trustworthy data. “You know that you are doing the best, to your knowledge,” she says, “because experts are providing you with guidance of what things you need to consider.”
Yet oddly among the growing number of research-
ers who are using dPCR in their experiments, Alikian is in the minority. Since publication in 2013, the digital MIQE guidelines have been cited 74 times, according to Google Scholar. PubMed lists over 400 dPCR papers in the same period. Similarly, the original MIQE guide-
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