microRNAs in Asthma miRCURY LNA™ Universal RT microRNA PCR
Dr. Stefan Dehmel
Dr. Katrin Milger
Dr. Stefan Dehmel
and Dr. Katrin Milger
study early origins of asthma in the Research group of PD Dr. Susanne Krauss-Etschmann at the Comprehensive Pneumology Center (LMU) in Munich, Germany. They have been using Exiqon's miRCURY LNA™ Universal RT microRNA PCR system to profile microRNAs in mouse serum in order to identify microRNA based biomarkers for asthma.
1. What is the main focus of your research and how did you come to be interested in microRNAs?
Our lab is interested to understand the underlying mechanisms of how early life exposures affect later asthma risk. Epidemiological studies have shown that prenatal and/or early postnatal exposures strongly influence the risk to develop asthma later in life. We hypothesize that microRNAs are potentially important mediators of this early programming and transgenerational transmission of disease risk.
2. What is the aim of your current project?
Asthma is difficult to diagnose in children below 5 years of age because lung function testing is difficult in this age group and further, because asthma-like symptoms can be provoked by respiratory tract viral infections. However, early asthma diagnosis is important to initiate appropriate treatment. The current project aimed to identify microRNA-based biomarkers for asthma in serum. We performed this initial study in a well-characterized murine model of asthma induced by ovalbumin.
3. How did you perform the experiments and analyze the results?
We used the Exiqon Serum/Plasma Focus Panels to identify miRs with robust expression in serum and found that most of the miRs that are conserved between mouse and human could be measured in our sample. For the subsequent analysis we used the GenEx software, which provides all the necessary tools, and applied global mean normalization.
4. What were some specific challenges in your experiments?
Changes in sample composition especially cell lysis can significantly alter the miR composition of serum and plasma. Further PCR/RT inhibitors are present in serum, and contamination in the extracted RNA may lead to varying efficiencies of the qPCR assays.
5. How did you overcome them?
We implemented several quality control steps including photometric analysis of serum, analysis of RNA by bioanalyzer and RT/PCR efficiency testing, choosing only the best quality samples for further analysis.
6. How do you feel about your results so far?
We are confident about our results and could confirm microRNA regulations in an independent subset of samples.
7. What do you find to be the main benefits/advantage of the Exiqon LNA™ microRNA qPCR system?
High sensitivity is a great advantage of the Exiqon LNA™ microRNA qPCR system enabling us to study microRNA expression in minimal amounts of starting material, like in our case 50µl of mouse serum. A further benefit of the Exiqon system compared to other qPCR based systems is the Universal RT reaction allowing analysis of all microRNAs from just one RT reaction. This, as well as the absence of a preamplification step, makes this technique more reliable and reproducible while saving time and money at the same time.
8. What would be your advice to colleagues about getting started with microRNA analysis?
It is advisable to take some time in the beginning to optimize sample preparation and RNA extraction for your specific application in order to get the most information out of your material and ensure that your results are reliable and reproducible.
9. What are the next steps in the current project and how do you plan to perform them?
To identify a robust and disease specific biomarker signature, further studies in mouse models using different allergens to induce asthma are planned. Furthermore, we plan to test a set of pre-identified microRNAs in human serum samples of a well characterized longitudinal pediatric cohort.
10. When and where will be heard /read more about your studies?
We are currently preparing a manuscript for publication.