The role of long non-coding RNAs in the heart lncRNA silencing using LNA™ GapmeRs
Introduction Rudi Micheletti
works at the Department of Medicine, CHUV (Centre Hospitalier Universitaire Vaudois in the laboratory of Thierry Pedrazzini) in Switzerland. He has been using Exiqon’s LNA™ GapmeRs to identify and characterize cardiac specific lncRNAs.
What is the general focus of the research conducted in your lab?
My PhD-project is mainly focused on characterizing the role of long non-coding RNAs in the heart. The research in my lab focuses on studying the key molecular mechanisms driving the maladaptive response of the stressed heart such as cardiac regeneration, fibrosis and inflammation.
What is the background for your current project involving LNA™ GapmeRs?
We tested Exiqon’s LNA™ GapmeRs in different cell types such as cardiomyocytes and cardiac fibroblasts with the aim of knocking down specific long non-coding RNAs.
What, if any, was your previous experience with RNA silencing?
In previous project we have used virus transduction carrying a short hairpin RNA (shRNA) designed to specifically target our candidate lncRNA. In our hands, we struggled to get an efficient knock down using shRNAs. The efficiency of transduction was quite low even at high MOI (multiplicity of infection) and we had to select the infected cells using proper antibiotics for several days. We tested 3 different shRNAs against each of our 4 candidates and we had a knock down greater than 50% just with 2 shRNAs against one of the candidates. We decided to move on with GapmeRs due to the problems just described.
What is the specific aim of your current project?
The aim of my project is to identify cardiac specific lncRNAs able to modulate the response of the heart to stress. We think lncRNAs play an important role in the infarcted heart by controlling the expression of important protein coding genes involved in cardiac remodeling.
How did you perform the experiments and analyze the results?
By using the on-line tool on Exiqon website, we designed Gapmers against 35 different lncRNAs (one GapmeR against every candidate). The online tool is user friendly and it gives you a list of GapmeRs against your target really quickly. We then isolated cardiomyocytes and cardiac fibroblasts, neonatal mouse hearts by digestion with proper enzymes mixes. The isolated cells were seeded on gelatinized plates at the correct confluence and transfected with 50nM of GapmeR (or scrambled) using a canonical transfection reagent. 48h after transfection, RNA was isolated and the knockdown efficiency was checked by measuring the level of target RNA by qRT-PCR.
What were some specific challenges in your experiments?
Mouse cardiomyocytes are cells quite fragile and difficult to isolate and they suffer upon transfection. We were afraid about aspecific effects of GapmeRs in this cell type.
How did you overcome them?
Cardiomyocytes were transfected using different concentration of GapmeRs. We didn’t observe any toxic effects with scrambled transfected cells and they didn’t show any change in commonly used cardiac stress markers.
How do you feel about your results so far?
I feel confident about my results. GapmeR knock down was reproducible and specific to my target. The percentage of GapmeRs able to knock down the specific target was impressively high. 85% of tested GapmeRs (one gapmer per target) provided knock down greater than 60%!
What do you find to be the main advantage of the LNA™ GapmeRs from Exiqon?
The design, the transfection and the experiment per se are easy to plan and to perform. The efficiency of the knockdown obtained is greater than methods used in the past and results are more reliable because I could reproduce them multiple times.
Why would you recommend Exiqon and/or Exiqon’s LNA™ GapmeRs to colleagues?
I will recommend Gapmers to my colleagues for all the advantages described above. Moreover, I didn’t find any specific problem using GapmeRs.
What would be your advice to colleagues about getting started with RNA inhibition experiments?
Carefully optimize as much as possible. I will recommend testing different time points and different concentrations of GapmeRs to find the optimal conditions.
In your opinion what is the most important factor for a successful gene silencing experiment?
GapmeRs concentration and time of transfection
What are the next steps in the current project and how do you plan to perform them?
I want to test the impact of modulating the expression of my targets in vivo by administration of GapmeRs in a mouse cardiac pathological model.