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Application Stories

Can microRNAs tell you how fit you are?

microRNA PCR Services
Dr. Anja Bye
Dr. Anja Bye
Dr. Anja Bye works at the K.G. Jebsen Center of Exercise in Medicine at the Norwegian University of Science and Technology (Trondheim, Norway) where they seek to combine epidemiological, experimental and clinical research into an integrated approach in the battle against lifestyle-related disease. Her recent publication (PLoS ONE 8(2): e57496) describes the use of microRNA profiling to identify novel markers for aerobic fitness, an important risk factor for cardiovascular disease.

What is the main focus of the research conducted in your lab?

Our research focuses on identifying the key cellular and molecular mechanisms underlying the beneficial effects of physical exercise on the heart, arteries and skeletal muscle in the context of disease prevention and management through experimental, clinical and epidemiological studies. Identifying the cellular and molecular mechanisms associated with aerobic fitness is important, because it may help us develop new and better methods to prevent and treat cardiovascular disease.

How did you come to be interested in microRNAs?

I read several papers on the topic, and I think there is a large potential of the circulating microRNAs as early markers of disease. It is particularly interesting that new miRs are still being discovered, and allows for more future biomarkers.

What was the specific aim of this project?

Aerobic fitness measured as maximal oxygen uptake (VO2max) is a good indicator of cardiovascular health, and a strong predictor of cardiovascular mortality in healthy individuals and in patients with cardiovascular disease (CVD). As low aerobic fitness is an important risk factor of CVD, microRNAs that are upregulated in subjects with low aerobic fitness may represent early biomarkers of CVD. The aim of this study was to assess whether circulating microRNAs (miRs) are associated with VO2max-level in healthy individuals.

What, if any, was your previous experience with microRNA analysis?

We had no experience in microRNA analysis and therefore looked to Exiqon Services to help us with the microRNA profiling.

How did you design the study?

The samples used in the study were part of a large health study carried out in Norway between 2006 and 2008 (The HUNT fitness study). The samples chosen for this biomarker discovery project had to be from participants that were matched on a lot of criteria such as age, gender, levels of physical activity, BMI, blood pressure as well as serum levels of glucose, cholesterol and triglycerides. Thus, the only cardiovascular risk factor that should be different between the cases and the controls was VO2max.

In the screening study we profiled 720 microRNAs in serum samples from matched healthy individuals (40-45yrs) with high (n=12) or low (n=12) VO2max . Candidate microRNAs were validated in a second cohort of subjects with high (n=38) or low (n=38) VO2max.

How did you perform the experiments and analyze the results?

We submitted serum samples to Exiqon Services who isolated the RNA and performed the microRNA profiling experiments. The RNA quality was controlled using RNA spike-ins. The screening study was performed on microRNA Ready-to-Use PCR, Human panel I and panel II.

Based on the screening approach, the 8 microRNAs were selected for further testing in the validation cohort. Additionally, three microRNAs that were stably expressed in the screening cohort were also included in the validation cohort as candidate reference genes. These 11 microRNAs were profiled using Pick-&-Mix panels.

After pre-processing, data was normalized using either global mean (in the screening study) or miR-425 as a reference gene (in the validation study). The data was then analyzed to find correlations between microRNA levels and fitness indicators as well as other CVD risk factors. Our main finding was that miR-210 showed a significant inverse correlation with VO2max.

What were some specific challenges in this project?

There were a couple of challenges associated with making sure the individuals included in the study were as similar as possible apart from their VO2max score. For instance, the analyzed blood samples were collected from non-fasting individuals. However, the time since last meal was similar in both the high and low VO2max group (3.1 hours). We therefore believe that food intake did not influence the miR results. Another limitation of the study is that 8 of the 100 participants reported to have performed exercise training the same day as blood sampling, spanning from 1 hour to 11 hours before the samples were collected. Baggish et al have previously shown that exercise training <24 hours before blood sampling can influence the levels of circulating miRs such as miR-222 and miR-21 independent of VO2max. However, removing these 8 participants from the statistical tests had no influence on the results.

How do you feel about your results so far?

We feel that we can trust the results, especially for the findings of the association between miR-210 and aerobic capacity. We are planning to follow-up this microRNA as an early marker of cardiovascular disease.

What were some important aspects for you when choosing a microRNA service partner?

Price is always an important consideration, but we were also very conscious about the quality of the analysis and the content of the final report. It was essential to us that the RNA-isolation service was available and that we could get our results within a short time frame. Finally, it was important that the service provider had a good reputation.

Why would you recommend Exiqon Services to colleagues?

I would definitely recommend Exiqon Service if you want a high quality comprehensive service at the right price.

What would be your advice to colleagues about getting started with microRNA profiling and biomarker discovery?

Make sure you are able to get high quality samples from a well described patient population and be sure to make the right group stratifications.

In your opinion what is the most important factor for a successful microRNA profiling experiment?

That you can feel sure about the technology used to measure the microRNAs and that you obtain a good normalization and find the most appropriate housekeeping miRs (for endogenous normalization). The last one is a big challenge these days.

What are the future perspectives for this research?

We are hoping to contribute to an early identification of cardiovascular disease, and to be able to complement the current clinically used markers for CVD.

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