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Identifying new prognostic markers and treatment approaches for thrombosis

mRNA silencing using LNA™ GapmeRs

Dr. Eugenia de la Morena-Barrio
Dr. Eugenia de la Morena-Barrio is a postdoc working in the laboratory led by Prof. Dr. Vicente Vicente García at the Department of Internal Medicine, University of Murcia, Spain. The work of this group is focused on Thrombosis and Haemostasis.

What is the main focus of your research?

Our main goal is the identification of new mechanisms involved in the risk of thrombosis. For that purpose, we study patients with idiopathic thrombosis, antithrombin deficiency and family history of thrombosis. Our research is mainly translational. The final objective of our research is to translate the results obtained in the laboratory on the molecular and biochemical basis of thromboembolic disorders to the benefit of patients, developing new diagnostic tools, identifying new prognostic markers and developing new treatment approaches.

How did you become interested in using GapmeRs for mRNA knockdown?

We have clinical and laboratory data from patients with thrombosis suggesting novel candidate genes involved in the risk of developing thrombosis. In order to assess these genes in human cellular models we needed to knock down their expression, however we failed to achieve this with classical silencing approaches. For this reason we turned to GapmeRs and these have been working well in our hands.

What is the specific aim of your current project?

The current project aims to find new molecular mechanisms responsible for antithrombin deficiency in patients with unknown underlying genetic defects.We would like to find out if our candidate genes are involved in the regulation of antithrombin levels by in vitro silencing with gapmers.

Did you try other methods of gene silencing prior to this?

Yes, I have used other type of silencers, mainly siRNA from other companies with poor results for the candidate genes and with certain cell lines, like HepG2.

How did you perform the experiment and analyze the results?

The silencing experiments were carried out in two different human hepatic cell lines, HepG2 and PLC/PRF/5.

The protocol I used is the following:

Plate 80.000 cell/well in a 24-well plate
  • The day after, remove the culture medium and transfect the cells
  • Transfecting agent: SiPORT™
  • Culture medium: OptiMEM
Procedure per well:
  • GapmeRs 100nM final concetration (I also tried 25nM, 50nM but the results were not as good). I have tried 5 different GapmeRs for a single target. They all achieved good target knockdown, although two of them were slightly more potent
  • GapmeRs and 1ul of transfecting agent were mixed well and incubated in 50ul of OptiMEM, 15min at room temperature
  • 450ul of OptiMEM was added and 500ul of this mixture was put into the well. It is crucial to do it very softly, drop by drop and to the wall of the well, never directly to the cell
  • 12 h post-transfection, I replaced the OptiMEM medium adding CDCHO serum free medium for another 24h. (I also tried to harvest cells after 12 and 48h but with worse results). The serum free medium appeared to improve transfection somewhat, but was primarily nescessary because I needed to evaluate secreted proteins that are also present in serum
I observed between 80-90% silencing of my target in PLC cells and 65-75% silencing in HepG2. I consider that these differences are caused mainly because HepG2 are difficult to transfect rather than a matter of gene expression which is similar in both cell lines. As a control, I used a negative silencer GapmeR provided also from Exiqon.

The silencing determination was performed by:
  • RNA extraction using RNAzol and following the manufacturer protocol
  • AppropriatecDNA generation
  • Relative quantification by qRT-PCR using beta actin as the reference gene and the convenient Taqman probes
I also tried the‘Reverse transfection protocol’ described in the SiPORT™ Amine Transfection Agent instruction manual but I obtained very poor cell viability.

Were there any specific challenges in this project?

Initially I used siRNA to knockdown my target, but I was unable to achieve more than 70% reduction in mRNA leves which was insufficient for my purpose. Once I started using GapmeRs I only had to optimize GapmeR concentration, time of incubation and cell density and then I observed close to complete knockdown of my target.

How do you feel about your results so far?

I am really happy with the degree of silencing I have obtained with GapmeRs technology. I would recommend this technology to those who work with cell lines that are not easy to transfect or those who need a high degree of knockdown of their target gene.

What do you find to be the main benefits of the LNA™ GapmeRs from Exiqon?

The main benefit of Exiqon LNA™ GapmeRs is that you can obtain a very high silencing degree of your interest gene and that this silencing is quite reproducible in different cell lines.

In your opinion what is the most important factor for a successful gene silencing experiment?

From my experience I think that the most important factors are:
  • The appropriate silencer technology
  • The silencing agent
  • The cell line. It is very important to optimize cell density for each cell line. It is also important to do the seeding when the cells are in exponential phase and with the less passage numbers possible after having thawed them. Postranslational modifications of proteins are highly sensitive to the number of cell passages
  • The handling. Critical to avoid cell mortality is the way the transfecting mixture is added to the cells. It is crucial to do it drop by drop and along the wall of the wells
  • Avoid using antibiotics in the transfection medium and we took care to avoid presence of phenol red in the medium as it inhibits estrogen receptors

What are the next steps in the current project and how do you plan to perform them?

The next steps will be to evaluate the levels and biochemical features of antithrombin released to the conditioned medium in cell lines where candidate genes where efficiently silenced with Exiqon GapmeRs.

What are the future perspectives for this research?

We hope that analysis of our in vitro GapmeR knockdown experiments will confirm our data obtained from patients. Collectively these new data might reveal new thrombophilic proteins and mutations involved in regulation of antithrombin levels and function.

When and where will be hear/read more about your studies?

These studies are now in preparation for publication. We estimate that they will be published sometime during 2015.


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