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Application Stories

MicroRNAs in Muscle Cell Differentiation

miRCURY LNA™ microRNA In Situ Detection Probes

Dr. Dylan Sweetman

Dr. Dylan Sweetman is working within the group of Dr. Andrea Münsterberg at the University of East Anglia School of Biological Sciences. The group investigates cellular and molecular mechanisms that underlie embryonic development.

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

We use the chick embryo as a vertebrate model organism. We are particularly interested in the signals that control cell fate decisions. For example, we are looking at developing somites, which give rise to skeletal muscle, cartilage, bone and tendon, and we have characterized the origin of cells that generate the vertebrate heart. Our work has provided insight into the mechanisms that are used to specify these different cell types and tissues. In particular, I am investigating the function of microRNAs in skeletal muscle differentiation.

2. What made you want to study miRNAs?

We decided to investigate microRNAs primarily because of the excellent work of Ronald Plasterk’s group looking at microRNA expression patterns in zebrafish by in situ hybridization (Science. 2005 Jul 8;309(5732):310-1). It was clear from this study that some microRNAs show interesting muscle-specific expression patterns.

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

I have several years experience in mRNA in situ hybridization using traditional RNA antisense probes on whole mount samples from a variety of organisms, e.g. chicken, mouse and Drosophila .

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

Basically no other technology has been demonstrated to work for microRNA detection in situ . Following the success of LNA probes as demonstrated in Plasterk’s lab, we decided to take the same approach.

5. What label and detection method did you choose?

I have tried end labeling the LNA™probe myself using DIG-UTP, but I found that the staining took several weeks to develop. That is why I started using the LNA™ probes pre-labeled with 5’- and 3’-DIG. The double DIG labeled probes give a much stronger signal, so staining requires a much shorter time. As well as the time saving, the double DIG probes usually result in a higher signal to noise ratio, although extensive washes with high detergent TBST buffer are also very important for removing background in my experience ( see protocol ).

6. What were the main challenges to develop a protocol for microRNA detection in your samples, and how did you overcome them?

Getting the technique working did require a certain amount of optimization from the original protocol. This involved mainly choosing appropriate temperatures for the hybridization and washing steps. I found that the temperatures need to be optimized for each probe, using 21C below the estimated melting temperature as a starting point. You also have to be quite patient when waiting for the staining to develop!

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

In general, I think that the specific labeling patterns that we observe are the best kind of a positive control. As you can see from the images we see very little background. We know that the labeling we observe is specific, because we see a different expression pattern for miR-1 and miR-206, which are highly related microRNAs (only 2nt difference between the mature microRNA sequences). In spite of the high degree of sequence similarity between the two microRNAs, it is evident from the in situ images that miR-1 is expressed in heart, whereas miR-206 is not. If I was setting up the technique from the beginning, I would recommend using a strongly expressed microRNA such as miR-206 or miR-124 that has a tissue specific expression pattern (rather than a widespread expression pattern) as a positive control. As a negative control we have chosen to use a probe designed to detect a plant microRNA.

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

The results we have obtained using the LNA detection probes are excellent, and we have been able to publish the work (Dev. Biol. 2008 Jun 21) so we are very happy!

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

We plan to perform some Northern blotting of microRNAs in cell lines. We would also like to move on to study the function of these microRNAs, by introducing microRNA mimics and inhibitors by electroporation into somites.

10. Currently, what is the biggest challenge in your microRNA research?

The major challenge right now is to perform functional studies in vivo in the embryo using gain and loss of function approaches. We have been trying to construct a microRNA overexpression vector, but unfortunately this has proved quite time consuming to set up, and the correct processing of the microRNA should always be confirmed to make sure the mature product is generated.

Figure 1 Chicken and Mouse In Situ Hybridization  
Expression of muscle specific microRNAs during myogenesis.
Wholemount in situ hybridization using double DIG labeled miRCURY LNA detection probes in embryos of chicken (HH stage 20) and mouse (stage E 10.5) showing differential expression of miR-1 and miR-206. Arrowheads indicate somites (s) and heart (h).

(Click image learn more)

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