Identifying microRNAs involved in asthmamicroRNA Array Services
The Sanak lab: Drs. Sanak, Kaczor, Plutecka, Dziedzina and Jakiela
, MD, PhD. works at the Division of Molecular Biology and Clinical Genetics, Jagiellonian University Medical College, in Krakow, Poland. His lab has been using Exiqon's microRNA Array Services to identify microRNAs involved in asthma.
What is the main focus the research conducted in your lab?
We are investigating phenotypes of bronchial asthma. As a part of this study, we use a model of air-liquid cultures of fully differentiated human bronchial epithelial cells from asthmatics and controls. The cultures are studied for response to TH2 cytokines and rhinovirus infections on the level of gene expression and production of lipid mediators. We wondered if some of these responses could be orchestrated by microRNAs.
How did you come to be interested in microRNAs?
microRNAs have emerged as very promising biomarkers. We are however, more interested in the epigenetic regulation of differentiation in the respiratory epithelium, e.g. ciliated vs. goblet cells and propagation of the anti-viral innate response. It seems, that the respiratory epithelium, which is an active barrier of the airways, may not only respond with release of cytokines or prostaglandins, but there may be also microRNA signaling involved.
What was the specific aim of the microRNA profiling studies?
The first step was to characterize intracellular microRNA species in the epithelial cell culture, and to find differences in their abundance across experimental setups: infected vs. non-infected, asthmatic vs. non-asthmatic, metaplastic vs. normal.
What, if any, was your previous experience with microRNA expression analysis/profiling?
microRNA studies was a terra nova for us, although, we are doing a lot qRT-PCR experiments or genotyping/sequencing studies.
How were the experiments performed?
We completed a screening phase using Exiqon’s microRNA Array Service to generate a list of several intracellular microRNAs that showed a differential expression across tested conditions of the cell cultures. We are now carefully selecting candidates for a validation phase, in which individual microRNAs will be quantitatively compared using a much larger number of samples and including microRNAs released to the medium.
What were some specific challenges in your project?
The first challenge was a limited amount of total RNA from the cell cultures. Epithelial air-liquid cultures are maintained for 3-5 weeks in 12-well plate inserts. The collected material is used mostly for targeted expression studies using qRT-PCR. We did not plan separate experiments to screen for any microRNAs of interest originally.
Also, as mentioned before, we did not have any experience in working with microRNA profiling.
How did you overcome them?
We did not face any problems with the quantity or quality of material used for profiling experiments. It seemed that our methods routinely used for qRT-PCR were valid for microRNA arrays as well.
Assistance from Exiqon Services was invaluable in cross-checking of RNA integrity and purity. Perhaps even more important was their advice on appropriate selection of controls and statistical evaluation of results.
How do you feel about your results from the microRNA array profiling experiments?
These experiments were successful and we feel confident about the results. In the nearest future, perhaps another run of microRNA Array profiling will be completed using clinical samples that we are currently collecting.
What were some important aspects for choosing to perform microarray profiling in a Service lab?
In my opinion, if a laboratory is not using microarray techniques or is not equipped with the required instrumentation, it this is very a good choice to benefit from the experience and credibility of a professional service such as Exiqon Services.
From a practical point of view it seemed more convenient and affordable to us to separate screening/profiling from the qRT-PCR Validation. This screening phase resembles much typical expression microarrays, which benefits and drawbacks are well known, alike the recommended statistical interpretation of results. The microRNA Array profiling report gave straightforward suggestions regarding which microRNAs to select as candidates for validation. qRT-PCR is a technique used by us frequently so we can complete the validation using our own equipment.
What would be your advice to colleagues about getting started with microRNA profiling today?
It is beneficial to split a study into profiling and validation, and to benefit from the Exiqon services to conduct the experimental part that one does not have the possibility to do. We would therefore recommend this strategy to labs that are not equipped with microRNA array equipment or knowhow. This recommendation is supported by our positive experience with the study we have conducted in Exiqon Services.
What are the next steps in the current project and how do you plan to perform them?
To integrate the findings on microRNA profiles with our other results will require validation and perhaps experiments using transfection with vectors encoding some candidate synthetic hairpin microRNAs. It is also tempting to characterize pathways affected by microRNAs using in silico prediction followed by mRNA and immunochemistry measurements.
What are the future perspectives for this research?
There is currently a limitation on interpretation of microRNAs data using systems biology. We collaborate with a team of skillful bioinformaticians, to complete unbiased prediction of mRNA targets for microRNAs. However, this approach will meet our expectations only if the predicted targets fit into meaningful cellular pathways.
When will we hear about your results?
Our microRNA study is ongoing, but for readers interested in our experimental model a recent publication is cited (Jakiela et al.
Prostaglandins Other Lipid Mediat 2003. PMID: 23742951