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Application Stories

MicroRNAs in Arteriogenesis

miRCURY LNA™ microRNA Detection Probes

Kerstin Troidl
Dr. Kerstin Troidl
Dr. Kerstin Troidl at Max-Planck Institute in Bad Nauheim, Germany, studies microRNAs involved in the growth of collateral arteries. She has produced some amazing ISH images of muscle cryosections using miRCURY LNA™ microRNA Detection Probes.

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

Our research focuses on fluid shear-stress induced vascular remodeling and we are in particular interested in the growth of collateral arteries, a process termed “arteriogenesis”. In a rat model of ligature of the femoral artery we surgically increased the collateral flow by an arterio-venous anastomosis distal to the ligature. This led to highly elevated fluid shear stress and thereby to an extreme arteriogenic response.

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

We used this strongly stimulated collateral tissue for molecular analyses. In a microRNA screening of these fluid shear stress exposed growing blood vessels in comparison to quiescent vessels we found differentially expressed miRNAs.

3. What is your previous experience in  in situ hybridization?

We were experienced in mRNA in situ hybridization using DIG labeled probes and combined our established protocol with the Exiqon protocol for microRNA ISH.

4. Why did you choose to use the miRCURY LNA™ microRNA detection probes from Exiqon?

The LNA technology seemed to be convincing and there were detailed protocols available.

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

Most of the double DIG labeled LNA probes worked immediately and resulted in impressing micrographs, which allowed us to localize the vascular miRNAs on a cellular level. We succeeded in colorimetric detection as well as in fluorescent detection and were able to combine ISH with immunostainings.

6. What positive and negative controls do you feel are important for in situ hybridization experiments?

Scramble-miR probes are essential negative controls and a well known smooth muscle cell specific miRNA (miR-145) served as positive control.

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

The Obernosterer protocol is a good starting point for ISH on rat tissues, but be aware, that your particular tissue might need a specification or adaption. We have for example added an 10 min Glycine ( 2mg/ml) step to efficiently stop Proteinase K treatment (from our conventional in situ protocol).

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

The high product quality, detailed protocols as well as individual and personal communication were essential prerequisites for choosing Exiqon as a supplier.

9. What were some specific challenges in your project and how did you overcome them?

The main challenge was the combined microRNA ISH and immunostaining experiments. In this case we found that we had to reduce the proteinase K treatment in order to get the best results.

10. Will you go on to validate your in situ hybridization results using other techniques (qRT-PCR, Northern blot etc.)?

Yes, we quantified miRNAs by qRT-PCR in order to validate our in situ hybridization results. For functional experiments to locally modulate miRNAs in arteries we have started to use exiqon's in vivo LNA inhibitors. In pilot experiments we were happy to recover the fluorescently labeled inhibitors in the collateral vessel wall.


Additional information

Figure 1

Cryosections of rat adductor muscle tissue. (Click to learn more)
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