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Application Stories

MicroRNAs in Skin

miRCURY LNA™ microRNA Detection Probes

Dr. Liming Luan
Dr. Liming Luan at Vanderbilt University Medical Center in Nashville studies the role of microRNAs in normal and pathological conditions of the skin. Here, he presents a new EDC-based in situ hybridization protocol.

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

To better define the role of miR-31 in squamous epithelial biology and keratinocyte proliferation in vivo , we generated doxycycline-inducible K14-rtTA/tet-O-miR-31 double transgenic mice mimicking the over-expression of miR-31 in SCCs, wounds and premalignant lesions. I tested miR-31 expression in our transgenic mice with your miRCURY product, the LNA™ miR-31 probe.

2. Why did you choose to use miRCURY LNA™ miRNA detection probes from Exiqon?

My mentor, Dr. Thomas Andl, recommended Exiqon company to me. And we both know that Exiqon is one of the leading suppliers for miRNA-related products.

3. You provide a new EDC-based in situ hybridization protocol. What led you to develop this?

I’ve tried to detect miR31 many times – I tried frozen sections, FFPE sections, and several different protocols. It turned out the best results I can get is to follow the protocol I provided, which combines Pena’s protocol (Pena, et al. 2009), Dr. Wigard Kloosterman’s protocol, and my old mRNA ISH protocol (adapted from Molecular Cloning). All of these protocols are listed in the “References” part of “mine”.

4. How does your protocol differ from that of Pena et al. 2009?

Only the “EDC fixation” part (step 13-17) is the same with Pena’s protocol. All others, like prehybridization, hybridization, stringency washes, and detection, are different from it.

5. How do you feel about the in situ hybridization results you obtained?

After all the efforts, I am pretty happy with it.

6. What advice would you give to researchers who want to get started in miRNA in situ hybridization detection?

Start with one or two miRNA(s) that are abundantly expressed in your tissue/cell of interest. Different protocols might be suitable for different tissues/cells, so, be patient, and try more protocols.

7. What are your next steps in your microRNA research?

Functional analysis: I am doing the miR31 inhibition related studies right now, and will make or purchase a miR-31 precursor overexpression plasmid for further studies.

8. Where will your research be showcased next?

Part of our miR-31 related work has been presented in The 70th Annual Meeting of the Society for Investigative Dermatology. 2010. Atlanta, Georgia. But miR-31 in situ data was newly generated, and wasn’t presented in the meeting. We are preparing a manuscript, in which, I think, we will add this data. However, I am not sure yet which journal we will submit it to.


Additional information

Figure 1

miR-31 in K14-miR31 transgenic mouse (upper panel) and control (lower panel)
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