They can be collected from routinely collected fluids such as blood, saliva, and urine. However, a number of factors can interfere with the accuracy of miRNA tests. In a study published online today in the Journal of Molecular Diagnostics, a group of researchers provide clear procedures for the collection and analysis of miRNA, significantly improving their diagnostic accuracy.
"Our study demonstrates that inherent differences in biological samples and the methods used to collect and analyze them can dramatically affect the detection and quantitation of microRNAs," reports lead investigator Dominik M. Duelli, PhD, Department of Cellular and Molecular Pharmacology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. "We developed measures to overcome the interfering activities and improved the sensitivity of miRNA detection up to 30-fold."
Over 1,000 miRNAs exist in the human body. Deregulation of specific miRNAs is associated with disease. Measuring the amount of miRNAs in body fluids can aid in the diagnosis of disease or conditions such as pregnancy. Dr. Duelli and his colleagues quantified two miRNAs: miR-16, which acts as a tumor suppressor and is deregulated or lost in some cancers, including breast cancer; and miR-223, which has been implicated in pregnancy and other conditions, as well as in some malignant diseases.
"A fundamental challenge to making microRNA diagnostics broadly available has been the inability to isolate enough high quality material to analyze. Our paper outlines ways of effectively collecting blood plasma samples, thus bringing us one step closer to the goal of making [miRNA] disease diagnostics a reality," adds co-investigator Sarah Linnstaedt, PhD, of Duke University.
The authors found that the choice of blood collection tube affects quantitation. Traditional green-top heparin tubes interfered nearly completely with miRNA detection. Grey-top tubes containing the anticoagulant sodium fluoride and potassium oxalate (NaF/KOx) provided the best results. Although miR-16 is about 500 times more abundant in blood plasma than miR-223, the results for both were similar, indicating that the differences in detection resulting from the choice of collection method apply to other miRNAs. Furthermore, collection of miR-223 in serum yielded more variable results, signifying that for some miRNAs, analysis of blood in plasma form is preferred.
The study indicated that natural components of blood plasma co-purify with miRNAs, interfering with their detection. The authors identified extra steps in purification, and the ideal dilution level, to reduce the interference. "Although counterintuitive, by reducing the starting material, inhibitors were presumed to be diluted below a threshold of interference. Careful titration of starting material yields more accurate miRNA quantitation," explains Dr. Duelli. In another approach, the authors avoided the problem of contamination by combining an enzyme that overcomes plasma inhibitors with standard enzymes to increase the sensitivity of miRNA detection by about 30-fold.
Finally, the authors observed that differences in plasma composition among individual donors yield different miRNA measurements. "These results raise the possibility that factors including diet, exercise, circadian rhythms, and seasons, which alter the blood chemistry, might affect miRNA detection and quantitation," says Dr. Duelli.
"The implications of this work are that without consideration of the variables we have identified, miRNA quantitation of human samples may not be reliable for the purpose of biomarker development. We provide approaches that enable faithful quantitation of miRNA abundance in body fluid," concludes Dr. Duelli.
The article is "Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation," by D-J. Kim, S. Linnstaedt, J. Palma, J. Cheol Park, E. Ntrivalas, J.Y.H. Kwak-Kim, A. Gilman-Sachs, K. Beaman, M.L. Hastings, J.N. Martin, and D.M. Duelli (doi: 10.1016/j.jmoldx.2011.09.002). Published online ahead of its issue, the study will appear in the Journal of Molecular Diagnostics, Volume 14, Issue 1 (January 2012) published by Elsevier.
David Sampson | EurekAlert!
Microscope measures muscle weakness
16.11.2018 | Friedrich-Alexander-Universität Erlangen-Nürnberg
Good preparation is half the digestion
16.11.2018 | Max-Planck-Institut für Stoffwechselforschung
Researchers at the University of New Hampshire have captured a difficult-to-view singular event involving "magnetic reconnection"--the process by which sparse particles and energy around Earth collide producing a quick but mighty explosion--in the Earth's magnetotail, the magnetic environment that trails behind the planet.
Magnetic reconnection has remained a bit of a mystery to scientists. They know it exists and have documented the effects that the energy explosions can...
Biochips have been developed at TU Wien (Vienna), on which tissue can be produced and examined. This allows supplying the tissue with different substances in a very controlled way.
Cultivating human cells in the Petri dish is not a big challenge today. Producing artificial tissue, however, permeated by fine blood vessels, is a much more...
Faster and secure data communication: This is the goal of a new joint project involving physicists from the University of Würzburg. The German Federal Ministry of Education and Research funds the project with 14.8 million euro.
In our digital world data security and secure communication are becoming more and more important. Quantum communication is a promising approach to achieve...
On Saturday, 10 November 2018, the research icebreaker Polarstern will leave its homeport of Bremerhaven, bound for Cape Town, South Africa.
When choosing materials to make something, trade-offs need to be made between a host of properties, such as thickness, stiffness and weight. Depending on the application in question, finding just the right balance is the difference between success and failure
Now, a team of Penn Engineers has demonstrated a new material they call "nanocardboard," an ultrathin equivalent of corrugated paper cardboard. A square...
09.11.2018 | Event News
06.11.2018 | Event News
23.10.2018 | Event News
16.11.2018 | Health and Medicine
16.11.2018 | Life Sciences
16.11.2018 | Life Sciences