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Quantitation of microRNAs in Tumor Angiogenesis

miRCURY LNA™ microRNA PCR System

Dr. Dorina Veliceasa

1. What is the current research going on in your lab?

We are interested in detecting changes in microRNA expression in endothelial cells that would target genes involved in the regulation of angiogenesis in the context of a tumor environment.

2. How did your research lead you to the study of microRNAs?

We are studying the transcriptional regulation of angiogenesis and we are interested in the identification of targets of different transcription factors. The microRNA field, it’s such a hot topic. There are so many discoveries happening around the involvement of microRNAs in gene regulation. With over 800 annotated human microRNAs, and with the fact that each microRNA can have multiple targets within the cell, there are just so many areas to choose from.

3. What were the key factors for you in choosing a microRNA supplier and partner?

We started by looking at microRNA primer availability for the microRNAs we are interested in. Exiqon had the best primer coverage for the microRNAs we wanted to study.

4. What made you choose Exiqon?

We spent some time learning about LNA technology, and we had a presentation at Northwestern by some Exiqon scientists. We had read a lot of information on the website, but the human contact was really helpful for us in choosing to work with Exiqon.

5. What were some specific challenges in your project?

At first, we used a qPCR approach, so it was really important to find good endogenous controls. Unfortunately, none of the typical controls worked very well in endothelial cells. We found a housekeeping microRNA which could be used as a control species in our model. But generally, the qPCR system, and the technology itself, is very good. After our qPCR work, we used some LNA probes for Northern blot, which gave us very good results. We’ve also used the new RNA isolation kit, and that worked well also.

6. How did you overcome them?

Mostly just a lot of testing and analyzing. We had to test many, many microRNA sequences to find the right control. Even a seemingly small change – as little as one cycle or even half a cycle – can have a big impact on normalization. As it turns out, let7a, whose regulation frequently changes in cancer models, was surprisingly very consistent between our experimental and control models.

7. What advice would you give to researchers who want to get started in microRNA research?

It’s very important to have high quality RNA isolation. The Exiqon kit seems to be very good in our mammalian system (cells grown in vitro ). For RT-PCR, it’s crucial to include several controls for the PCR reaction. You need to have several experiments, duplicates and triplicates, RNA isolated from different experiments, etc., in order to get consistent data. It’s especially important to have replicates to validate your data when you have very fine changes in the microRNA expression levels.

8. What would you tell a colleague about why they should work with Exiqon?

The primer coverage from Exiqon is very good, and it is updated frequently. The technology is very good, and not limited to one method – using LNA allowed me to avoid radioactive readout on my Northern blots. Also, the technical support is very good at Exiqon. Since microRNA was a completely new field for me when we started on this work, we had many questions along the way, and the scientists at Exiqon were very, very supportive. The response time, by phone or email, was short, and this was very helpful. In addition, our sales representative is great. He made it easy for us to get started in our projects with Exiqon’s products.

9. Where will your research be showcased next? (Articles, conferences, posters, etc.)

We are currently preparing manuscripts to submit for publication. We will have a poster coming soon, and then an article to follow. 

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