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The Role of MicroRNAs in Kidney Development

MicroRNA Array Profiling Services

Dr. Roman-Ulrich Müller
Roman-Ulrich Müller works in the lab of Prof. Thomas Benzing, previously at the Renal Division, University of Freiburg and currently at the Department of Medicine IV,  University of Cologne, and the Kidney Research Center Cologne. The lab focuses on the functional analysis of disease-relevant genes and their role in signal transduction in genetic kidney diseases. As part of this work they are studying microRNA expression in kidney development.

1. What is your research area?

We are working on kidney development in mammals and the role of kidney specific genes in the pathogenesis of congenital kidney diseases, e.g., nephronophthisis and FSGS.

2. What is your previous experience in microRNA profiling?

Until now we have been using small RNA cloning methods and small RNA Northern Blotting to detect small RNAs in our samples and establish miRNA-profiles in kidney development.

3. Why did you choose to use the miRCURY  LNA™ Array microRNA profiling services from Exiqon?

All the methods we have been using so far are pretty time consuming and are not exactly suitable for high-throughput profiling. As a lot of mammalian miRNA-sequences have now been published in miRBase, we judged that using micro-array technology would be the most favorable approach for our miRNA-projects.

4. In your opinion what are the main advantages to using the miRCURY LNA™ Arrays compared to other types of microRNA array?

Bearing in mind that some miRNAs only differ from each other in a single position, one major concern regarding miRNA-array technologies is specificity. In addition, some tissue specific miRNAs that are highly interesting can only be discovered using very sensitive assays as they are not abundant. miRCURY LNA -arrays show important advantages compared to DNA-arrays both in terms of specificity and sensitivity as the modified nucleotides show a considerably higher mismatch penalty.

5. What do you feel are the main advantages of using a service provider, instead of performing experiments in your own lab?

In our lab we currently do not perform array hybridization and scanning ourselves and take advantage of service providers as to mRNA-arrays as well. Thus we did not want to establish the techniques and obtain the necessary reagents and machines ourselves, but decided to get started right away having Exiqon perform the necessary steps. Consequently we did not have to solve any of problems that always occur using new techniques as these had been well established at the Exiqon service facility.

6. How do you feel about the results you received from the microRNA profiling services?

The results show some differentially regulated miRNAs and a nice overlap of expression regarding miRNAs encoded by a common primary transcript. Even miRNAs with a high level of similarity could be distinguished well using these arrays. Even though correlation coefficients of several replicates did not reach the levels that can be reached in whole genome chips (which is mainly due to the number of spots), we feel very confident about the results we were able to retrieve.

7. How did you go on to validate the results from the miRNA arrays?

As our main approach for validation, we chose miRNA in situ hybridization in cell-culture, whole-mount and whole organ experiments using miRCURY LNA in situ probes. The resulting data strongly confirms the results from the miRCURY miRNA-arrays and shows that LNA in situ hybridization is a nice way of validating data from screening and profiling approaches, that in addition provides further information about the localization of miRNA-expression at a suborgan level. 

8. Can you disclose your near future research plans? Do you plan further miRNA arrays experiments, further validation , or functional analysis?

We are working on qPCR-approaches to further validate the miRCURY LNA array-data on a larger scale. In addition, we are going to compare the LNA -array data to expression data obtained from DNA-arrays to further examine the advantages of LNA -technologies. Regarding functional analyses, we will take a closer look at the localization of miRNAs in the kidney and on a subcellular level. Furthermore we are going to screen for mRNA-targets of the most abundant and differentially regulated miRNAs that we were able to identify and check for their function using sequence specific miRNA-inhibitors and  lentivirally mediated miRNA-overexpression.


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