(Press-News.org) Seattle, Wash. — September 1, 2013 — A study published online in Nature Methods today demonstrated that Droplet Digital PCR (ddPCR™) technology can be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum across different days, paving the way for further development of miRNA and other nucleic acids as circulating biomarkers.
"In the field of circulating microRNA diagnostics, droplet digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories," said Dr. Muneesh Tewari, Associate Member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.
Challenges in miRNA quantification
miRNAs are small regulatory RNA molecules with diverse cellular functions. The human genome may encode over 1,000 miRNAs, which could target about 60 percent of mammalian genes. Because they are abundant in many cell types, exist in highly stable extracellular forms, and may provide direct information about disease processes, they are being actively studied as blood-based biomarkers for cancer and other diseases.
Quantitative real-time PCR (qPCR) has been used for the analytical measurement of miRNAs in blood samples; however, researchers have found that qPCR measurements of miRNAs in serum or plasma display unacceptably high interday variability, undermining the use of miRNAs as reliable blood-based biomarkers. An approach that yields more dependable results has therefore been sought by researchers in this field.
Advantages of ddPCR for miRNA detection
Digital PCR has many advantages over qPCR including the ability to provide absolute quantification without a standard curve and robustness to variations in PCR efficiency across different samples and assays. These and other advantages are embodied in
Bio-Rad Laboratories' QX100™ Droplet Digital PCR (ddPCR™) system, which was introduced in 2011.
"We chose to use Bio-Rad's QX100 Droplet Digital PCR system because it was the first system on the market that could make digital PCR practical from a cost and throughput standpoint for routine use in the lab," said Dr. Tewari.
To assess the imprecision introduced by each workflow step — serial dilution preparation, reverse transcription (RT), and PCR technical replicates — Dr. Tewari and his team conducted nested analyses of ddPCR vs. qPCR on cDNA from a dilution series of six different synthetic miRNAs in both water and plasma on three separate days. In comparison to qPCR, the researchers found that ddPCR demonstrated greater precision (48–72% lower coefficients of variation) with respect to PCR-specific variation
Next, the team performed a side-by-side comparison of qPCR to ddPCR for detecting miRNAs in serum. They collected sera samples from 20 patients with advanced prostate cancer and 20 age-matched male controls and measured the abundance of miR-141, which has been shown to be a biomarker for advanced prostate cancer. Samples were analyzed by qPCR and ddPCR with individual dilution series replicates prepared on three different days. They found that ddPCR improved day-to-day reproducibility seven-fold relative to qPCR. It was also able to demonstrate differences between case vs. control specimens with much higher confidence than qPCR (p=0.0036 vs. p=0.1199).
"Droplet digital PCR will allow us to accurately follow serum microRNA biomarker concentrations over time during a patient's treatment course, something that has been nearly impossible to achieve with real-time PCR," he said.
INFORMATION:
About Fred Hutchinson Cancer Research Center
At Fred Hutchinson Cancer Research Center, home to three Nobel laureates, interdisciplinary teams of world-renowned scientists seek new and innovative ways to prevent, diagnose and treat cancer, HIV/AIDS and other life-threatening diseases. Fred Hutch's pioneering work in bone marrow transplantation led to the development of immunotherapy, which harnesses the power of the immune system to treat cancer with minimal side effects. An independent, nonprofit research institute based in Seattle, Fred Hutch houses the nation's first and largest cancer prevention research program, as well as the clinical coordinating center of the Women's Health Initiative and the international headquarters of the HIV Vaccine Trials Network. Private contributions are essential for enabling Fred Hutch scientists to explore novel research opportunities that lead to important medical breakthroughs. For more information visit http://www.fredhutch.org or follow Fred Hutch on Facebook, Twitter or YouTube.
About Bio-Rad
Bio-Rad Laboratories, Inc. (NYSE: BIO and BIOb) has been at the center of scientific discovery for more than 60 years, manufacturing and distributing a broad range of products for the life science research and clinical diagnostic markets. The company is renowned worldwide among hospitals, universities, major research institutions, as well as biotechnology and pharmaceutical companies for its commitment to quality and customer service. Founded in 1952, Bio-Rad is headquartered in Hercules, California, and serves more than 100,000 research and industry customers worldwide through its global network of operations. The company employs over 7,600 people globally and had revenues exceeding $2 billion in 2012. For more information, visit http://www.bio-rad.com.
END
STANFORD, Calif. — A new study by researchers at the Stanford University School of Medicine could help pinpoint ways to counter the effects of the antibiotics-driven depletion of friendly, gut-dwelling bacteria.
A number of intestinal pathogens can cause problems after antibiotic administration, said Justin Sonnenburg, PhD, assistant professor of microbiology and immunology and the senior author of the study, to be published online Sept. 1 in Nature. Graduate students Katharine Ng and Jessica Ferreyra shared lead authorship.
"Antibiotics open the door for these pathogens ...
Demystifying the chemical processes that create a wine's aroma, and the invaluable potential application of that understanding in winemaking, is the new objective of scientists in Uruguay who, with European partners, also recently sequenced the genome of the high-value Tannat grape, from which "the most healthy of red wines" are fermented.
Meanwhile, a quick, $1 test in development by researchers in Paraguay and Uruguay promises to reduce the economic and health burden of 3 million cases of syphilis in Latin America -- a disease readily treated and cured if diagnosed ...
A drug proven effective for treatment of adults and children with sickle cell anemia reduced hospitalizations and cut annual estimated medical costs by 21 percent for affected infants and toddlers, according to an analysis led by St. Jude Children's Research Hospital. The report appears today in the advance online edition of the journal Pediatrics.
The study is the largest ever focusing on the economic impact of the drug hydroxyurea in children with the inherited blood disorder. The result supports expanded use of the drug to extend the length and quality of life for ...
Hamilton, ON (September 2, 2013) – Despite living with the highest risk factors for heart disease, people in high income countries suffer less from serious cardiovascular disease, says an international study by the global PURE (Prospective Urban Rural Epidemiology ) collaboration and led by McMaster University researchers.
At the same time, the study found that people in low income countries, although living with fewer risk factors for heart disease, have a higher incidence of serious cardiovascular disease including death.
"These findings were a total surprise," ...
Amsterdam, The Netherlands – Monday 2 September 2013: Metabolically healthy women have the same cardiovascular disease risk regardless of their BMI, according to research presented at the ESC Congress today by Dr Søren Skøtt Andersen and Dr Michelle Schmiegelow from Denmark. The findings in more than 260,000 subjects suggest that obese women have a window of opportunity to lose weight and avoid developing a metabolic disorder, which would increase their CVD risk.
Dr Schmiegelow said: "Obesity and/or metabolic disorders (hypertensive disorders [hypertension, gestational ...
Amsterdam, The Netherlands – Monday 2 September 2013 : Pacemakers implanted for slow heart rhythm restore life expectancy to normal levels, reveals research presented at ESC Congress 2013 today by Dr Erik O. Udo from the Netherlands. The findings provide a new reference point for the prognosis of modern pacemaker patients.
Dr Udo said: "Previous studies describing the survival of pacemaker patients used data that is more than 20 years old and cannot be used anymore for patient counselling and benchmarking. There have been considerable changes in pacemaker technology ...
AMSTERDAM, The Netherlands, 2 September2013 – The search continues for an agent that increases high-density lipoprotein (HDL) and reduces arterial plaque, after the experimental apolipoprotein A1 (apoA1) inducer, RVX-208 failed to do so in the ApoA1 Synthesis Stimulation and Intravascular Ultrasound for Coronary Atheroma Regression Evaluation (ASSURE) study.
The lack of efficacy of RVX-208 is "disappointing and surprising, given promising earlier findings," noted lead investigator Stephen Nicholls MBBS, PhD, Deputy Director at the South Australian Health and Medical Research ...
AMSTERDAM, The Netherlands - A common drug that is used to treat high blood pressure in the general population has been found to significantly reduce a dangerous and frequently fatal cardiac problem in patients with Marfan syndrome.
Results of the COMPARE (COzaar in Marfan PAtients Reduces aortic Enlargement) study reveal that patients treated with losartan (Cozaar) had a significantly reduced rate of aortic enlargement after 3 years compared to patients who did not receive the treatment.
"Our study is the first large, prospective randomized study to assess the effects ...
AMSTERDAM, The Netherlands – Patients with type 2 diabetes and high cardiovascular risk due to recent acute coronary syndromes had similar rates of cardiovascular events when treated with the anti-diabetic agent alogliptin compared to placebo according to results of the Examination of Cardiovascular Outcomes with Alogliptin versus Standard of Care (EXAMINE) trial presented at the European Society of Cardiology Congress.
"Compared with placebo, treatment with alogliptin resulted in similar rates of the primary endpoint, which was a composite of cardiovascular death, myocardial ...
To win the war against weight gain, it turns out that every skirmish matters – as long as the physical activity puts your heart and lungs to work.
In a new study published today in the American Journal of Health Promotion, University of Utah researchers found that even brief episodes of physical activity that exceed a certain level of intensity can have as positive an effect on weight as does the current recommendation of 10 or more minutes at a time.
"What we learned is that for preventing weight gain, the intensity of the activity matters more than duration," says ...
