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miRNA Blood Diagnostics by Dr. Dominik M.Duelli
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New progress in the field of evaluating miRNAs in Circulation


Adittional to SomaGenics’ method for miRNA plasma measurement, other scientists are also working on new methods to measure the miRNA circulating in the plasma, especially those associated with certain pathological states. Dominik M. Duelli, Ph.D., assistant professor at Rosalind Franklin University of Medicine and Science, has been experimenting with circulating miRNAs, and he and his lab have evaluated the most accurate and reproducible methods for detecting and quantifying miRNA from plasma using qRT-PCR.

Dr. Duelli and his lab got the idea for testing these various parameters of miRNA profiling from inconsistencies in detecting certain miRNAs in plasma.

“When we first started our work, the original idea for a biomarker measured in blood plasma was that it should reflect what’s inside the malignancy, so an miRNA that’s high inside the cell should also be high outside the cell,” Dr. Duelli explained. “We profiled miRNAs in an array of cell-line cultures and saw that 60–70% of the miRNAs had the same profile outside and inside cells. But some miRNAs appeared to be exclusively released 90% or more out of the cell. Another category was miRNAs that were retained and were not released at all.”
Dr. Duelli tried to evaluate whether this was normal, a physiological phenomenon, or were the miRNAs simply not detectable based on their methodology and using that specific technology?
They began their work by measuring miR-16, which is highly abundant in plasma and miR-223 with a low abundance in plasma, in fresh plasma from 16 subjects to determine the optimal parameters needed for miRNA profiling. They noticed that anticoagulants like, for instance, citrate or KOx/NaF are a much better option than heparin for miRNA quantitation in plasma. Heparin has a habit of inhibiting RT and polymerases, and detecting the miRNA’s had been possible only if they diluted the heparin or treated raw plasma with heparinase.

Heparin, also known as unfractionated heparin, is a highly sulfated glycosaminoglycan, and widely used as an injectable anticoagulant. It also has the highest negative charge density of any known biological molecule. It is often used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines, and in recent times is used to prevent coagulation in plasma and blood samples used for diagnostics and lab-work.


Dr. Duelli and his lab tried different methods for RNA isolation and noticed that some reagents can selectively precipitate certain miRNAs, which can cause some miRNAs not to be detected at all in a sample. Instead, by using a silica membrane or beads for RNA extraction they can prevent polymerase inhibitors from co-purifying with miRNA’s. This methof provided better purity and yield.

In addition, Dr. Duelli says that using a Taq polymerase, Hemo KlenTaq, from New England Biolabs, can solve this problem because some of the more significant inhibitors cannot bind to this truncated polymerase. Simply by including Hemo KlenTaq in the reaction Dr. Duelli’s lab observed a 30-fold increase in miR-16 and miR-223 expression.
Dr. Duelli’s lab also used fluorescent SmartFlare RNA Detection Probes, EMD Millipore, which utilize a non enzymatic approach for fast and direct miRNA quantification in live samples. They were also able to detect and quantify miR-16 expression in plasma within minutes to one hour after venipuncture, he says, which greatly contributes to the effectiveness of the process.

Dr. Duelli’s lab was focused, among other things, on making this technology applicable in the clinical setting, allowing for a many fold increase in diagnostic methods, and bringing medicine one step closer not only to personal, individualized medicine, but also helping doctors “detect and prevent” disease, instead of just curing and managing the symptoms.

“You can measure miRNA levels right after chemotherapy and correlate it with tumor progression; if the tumor is shrinking, you can measure if these miRNAs go away. It comes at a cost to sensitivity because there is no amplification, but it can be used in plasma or other samples.”
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