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Hutch Lab Improves Protocol for qRT-PCR Analysis of microRNA Biomarkers from Blood

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By Ben Butkus

Researchers from the Fred Hutchinson Cancer Research Center have developed and published an improved protocol for using quantitative reverse transcription PCR to analyze circulating microRNA biomarkers in blood plasma and serum.

The scientists hope the improved protocol will better enable researchers in other labs to conduct research on using microRNAs as biomarkers for cancer detection, the study's lead author said this week.

And although the technique is currently for research-only use, it could be an important step toward developing a blood-based cancer detection method based on circulating microRNAs.

"Right now we're using this only for research purposes, but certainly the long-term goal of all this is developing clinical diagnostics," Muneesh Tewari, a faculty member at Hutchinson and lead author of the study, told PCR Insider this week. "How that plays out remains to be seen exactly."

The researchers developed the improved protocol after Tewari's group published a paper in the Proceedings of the National Academy of Sciences in 2008 that described how miRNAs originating from prostate cancer cells could be measured in blood plasma to distinguish patients with cancer from healthy subjects.

Tewari said that following the publication, a number of researchers contacted his lab to discuss how they might be able to replicate the experiment and optimize the isolation of microRNAs from blood plasma.

"I've gotten a lot of inquires about the details of the protocol, and we gave a pretty detailed protocol in the supplementary methods [of the PNAS paper], but since then in small ways it's evolved," Tewari said.

The goal then, of the most recent study, published online last month in the journal Methods, was to "enable other researchers who are doing various kinds of biomarker research and are considering using microRNAs as biomarkers to be able to implement the technology in their labs," Tewari said.

Tewari added that most of the modifications to the protocol were "relatively minor," but that in the context of measuring microRNAs in plasma, they represent a new approach.

Most of the modifications are in the up-front part of the protocol that deals with extracting and isolating microRNAs from plasma and serum. For instance, the researchers scaled up the ratio of denaturing agent used in relation to plasma or serum, both of which have very high protein contents.

They tested this modification using two commercially available kits: Ambion's MiRvana PARIS, in which the only modification was an additional organic extraction step; and Qiagen's miRNeasy kit, in which the modification involved using 10 volumes of Qiazol reagent to initially denature one volume of plasma or serum.

Although both protocols have proven effective, the researchers wrote, the Qiagen protocol yielded two- to three-fold more RNA.

The researchers also made minor modifications to PCR reaction volumes; and routinely added non-human (specifically C. elegans) synthetic microRNAs after initial denaturation of plasma or serum to serve as internal controls and correct for variability in microRNA extraction.

On the microRNA analysis end of the protocol, the researchers made fewer modifications, such as reducing the reaction volumes for conducting quantitative real-time PCR based on Applied Biosystems Taqman kits. Tewari and colleagues used a Tetrad2 Peltier Thermal Cycler from Bio-Rad to carry out reverse transcription reactions; and an Applied Biosystems 7900HT thermal cycler for real-time PCR.

Tewari stressed that other protocols for extracting and analyzing microRNAs from plasma or serum exist, and that "other vendors also have good products for this. By no means is this the result of a careful head-to-head comparison. This is just what we're doing, and it's working with a certain level of performance. And by no means is it a final protocol. I'm sure it will be continually evolving."

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