Purification steps can remove the confounding influence of cell-based miRNAs, allowing more robust measurement of circulating miRNAs, Affymetrix researchers reported recently in a study in PLoS One.
Using the company's GeneChip miRNA microarray, the team evaluated a variety of methods to reduce the presence of cellular miRNAs in blood samples in order to allow more accurate and reproducible analysis of circulating miRNA for potential predictive signatures.
Keith Jones, vice president of biological science at Affymetrix, told Gene Silencing News this week that the study was spurred by the company's desire to demonstrate novel applications of its products, specifically its GeneChip miRNA array in this case.
"We had this great technology in the miRNA microarray and I wanted to demonstrate novel utility of it, so I went about picking a project," he said. "We ended up pretty quickly focusing in on miRNAs … [because] they show both tissue and disease specificity, and [have] very nice stability in circulation, as well."
The team initially planned to look for cancer biomarkers in circulating miRNAs, but as they began the study they realized that “the existing literature was filled with inconsistent results and we speculated this might be due to varying levels of processing artifacts,” he said.
“When one gets blood, there are a lot hematopoietic cells, lymphocytes, and leukocytes floating around. Depending on processing, there could be varying levels of leakage of miRNA from those cells, which are then interfering with the real signal that you're looking at," he added.
"I think there are real signatures out there, but in looking at some of the studies you see non-completely overlapping sets of miRNAs that have been identified,” Jones said. “We wanted to provide best practices on how to minimize those procedural artifacts."
In the paper, the authors wrote that different fractionation procedures have "varying degrees of efficacy" and "can play a major role in impairing circulating miRNA signatures." According to Jones, the techniques used in Affymetrix's study should make examining circulating miRNAs easier, more reproducible, and more robust.
"The deeper we got into this, the deeper we realized we needed to solve this problem and we could be doing the community a service by providing some best practices," he said.
The researchers first separated plasma from 17 healthy male donors with an initial low speed centrifugation, isolating out three cellular fractions: red blood cells, white blood cells, and leukocytes.
They hypothesized that these cell types represent the major contributors of confounding cellular miRNA material in circulation.
"These miRNAs that reside in this component are potentially confounding, and therefore the most conservative approach is to look not at those, but instead focus on non-overlapping miRNAs," Jones said.
Using sequential purification steps and measuring for the presence of cellular miRNAs with Affymetrix's GeneChip miRNA array, the group was "able to identify that in the supernatant from the spin, there was a fair amount of both particulate matter and miRNAs that mapped back to these cellular components," said Jones.
"By performing an extra centrifugation step, we were able to pellet down the particulate matter and increase the percentage of miRNAs that did not map back to a leukocytic profile," he said. "We showed that with two centrifugations, one has a much cleaner preparation of non-overlapping miRNAs."
The team also found that additional purification steps did not do much to further increase the percentage of non-overlapping miRNAs. "Therefore, our recommendation is that one should stabilize the plasma as quickly as possible and perform two centrifugation steps in order to minimize confounding species without negatively impacting the bona fide circulating population of miRNAs," he said.
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Taken together, the study authors wrote, the results "provide a comprehensive map of the circulatory miRNA environment in normal individuals and provide compelling motivation to explore such landscapes in detail in pathological conditions."
According to Jones, the team also wanted to show a little more of the array's performance in the paper, but didn’t want to go as far as measuring disease biomarkers. Instead, they investigated differences in circulating miRNA expression between males and females, examining samples from eight male and 10 female donors.
"We asked, 'Are there gender-specific markers that are floating around in circulation and can we identify them using our procedure?' And the answer was, 'Yes we could,'" Jones said.
Four circulating miRNAs were differentially expressed in the two genders, all of them up-regulated in females compared to males, the study authors reported.
Because the Affymetrix team's initial goal was to identify miRNA disease markers, they've now moved on to do just that.
Jones said the team has already done an additional study, which it is now writing up for publication, but he said the company can't reveal details about the focus of the new research.
"That was always our goal and we've actually got some pretty exciting results," he said.
In the meantime, Jones said he thinks the practices the group showed in the paper should be a boon not just to Affymetrix moving forward with biomarker identification, but also to other researchers.
"I think we've identified a robust procedure for stably isolating miRNA out of circulation that has minimal confounding variables from the leukocytic population. By following this procedure, we should be able to, or the community should be able to, generate less variable, more robust biomarker signatures," he said.
Eventually, Jones said, such signatures would hopefully lead to an assay for diagnostic or prognostic evaluation.
"I think that’s not only our goal, but lots of other people’s goal as well. I’m excited to use our new GeneChip miRNA 2.0 array set to start looking more broadly across multiple diseases," he said.
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