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Researchers Prime qPCR


Some researchers have set their sights on a new clinical application, beyond diagnosing infectious diseases, for quantitative PCR: mitochondrial DNA analysis. While several groups have shown the utility of qPCR for the detection and quantification of disease-associated mtDNA signatures in the lab, translating that discovery-stage research into clinical diagnostics remains difficult.

According to Afshan Malik, the current protocol for measuring mtDNA relative to nuclear DNA using qPCR is part of the problem. Indeed, in attempting to replicate their own qPCR-based mitochondrial to nuclear DNA results last year, she and her colleagues at King's College London found "that there were a lot of inconsistencies," Malik says. "We even found that if different people tested the same samples on different days we'd get totally different results."

That, she says, was largely a product of problems with the procedure. By retracing her group's steps and referring to the papers from which the protocols and primers were derived, Malik uncovered an un-nerving potential source of error.

After running checks on the "well-used primers" her group had employed, she says she was surprised to find "that they were present in the nuclear genome, which meant they could have been co-amplifying something other than the mitochondrial genome. Then I started looking into it more, and was horrified to realize that most people were using mitochondrial primers that also co-amplify other nuclear pseudogenes." Using primers that are not specific to the mitochondrial genome means that "the methodology is not actually measuring what people think it is," she adds.

While the potential for inaccurate mtDNA quantification "really depends on what region of the mitochondrial genome the primers are designed to," Malik says that, in general, using non-specific primers most often leads to "an overestimation of mitochondrial DNA content."

In a Biochemical and Biophysical Research Communications paper published online in June, Malik and her colleagues identified three potential sources of error in current qPCR-based mtDNA analysis protocols and proposed three matched solutions. Beyond the primer specificity problem, inconsistencies in template preparation, storage, and dilution approaches can also skew results, the researchers noted.

"We've suggested some steps to try and remove the inconsistencies," Malik says of her team's paper, in which they proposed modifications to the qPCR-based mtDNA analysis method. A standardized protocol to accurately and consistently quantify mtDNA in human samples could edge the approach into the clinic, where its application is increasingly desired — particularly as disease-associated alterations in mtDNA content and in mitochondrial function continue to pop up in the literature, Malik adds.

"There've been a lot of papers coming out ... in the last two or three years where people are measuring mitochondrial DNA in a very wide range of diseases, and in different body fluids, sometimes in disease versus control tissues," she says.

Malik says mtDNA could be a predictor of mitochondrial dysfunction. "If that's true, then there's a whole load of diseases which are now known to be affected by mitochondrial dysfunction, including aging, various neurodegenerative diseases, cancers, and complications of HIV," she says. In that way, she adds, mtDNA "could be a really valuable biomarker, because it's non-invasive." While rigorous validation work remains to be done, "I think it's possible that you could offer a blood test to patients on a routine basis to monitor the health of their mitochondria," she says.

To apply such biomarkers in the future, Malik says protocol standardization is imperative. "What we've published goes towards standardizing, but there's still more to do before it's ready for the clinic," she says.

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