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Application Stories

The Role of MicroRNA in Homeostasis

miRCURY LNA™ microRNA Arrays

Dr. Martina Muckenthaler
Prof Martina Muckenthaler is Head of Molecular Medicine at the University of Heidelberg. Her current research areas include the role of iron in health and disease, haematological malignancies and molecular diagnostics. Here, she describes part of her research on microRNA and how she has been able to use the technology incorporated in miRCURY LNA Arrays in her work.

1. Can you describe the current research going on in your laboratory?

My research focus is on regulatory mechanisms and gene networks involved in iron homeostasis and related disorders. The research group is integrated within the Molecular Medicine Partnership Unit (MMPU), an interdisciplinary cooperation between the European Molecular Biology Laboratory (EMBL) and the Medical Faculty of the University of Heidelberg. Within this unit we perform translational research that addresses regulatory circuits, such as those involved in non-sense mediated decay, cystic fibrosis or cancer.

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

In recent years it became evident that miRNAs can be highly valuable as clinical markers for understanding molecular disease mechanisms and possibly for therapeutic intervention. miRNA response patterns are currently being evaluated for their role as biomarkers in acute childhood leukemia and breast cancer as well as for the understanding of regulatory processes involved in maintaining iron homeostasis.
We have a long-standing interest in post-transcriptional control mechanisms and regulatory networks involved in disease. Plus, we have access to clinical samples within the Department of Medicine and within the National Genome Network (NGFN-2), which funds our research. These two factors, added to our expertise in establishing specialized microarray platforms, made us decide to develop a sensitive array for genome-wide miRNA expression profiling (miChip).

3. For what reasons do you think there is so much current interest in microRNAs?

The discovery of miRNAs has revealed a new regulatory concept of gene expression. Several observations that could not be explained before by protein coding mRNAs can now be explained by mechanisms involving miRNAs. For example, miRNAs are frequently located within chromosomal regions that have been closely linked to cancer. These include minimal regions of amplifications or loss of heterozygosity, as well as chromosomal breakpoints. For example in the case of B-cell chronic lymphocytic leukemia (CLL) the 13q14 chromosomal region has long been scanned for the presence of oncogenes or tumor suppressor genes – without success. Recently, mir-15 and mir-16 were discovered within this chromosomal location and it turns out that these miRNAs play a role in enhancing apoptosis by decreasing the expression of the anti-apoptotic factor BCL-2.

In addition, experimental data indicates that cell-type specific miRNA expression remains largely unchanged in resulting tumors. It seems that tumors are better classified by miRNA expression than by mRNA expression patterns that are rather more complex. This opens the possibility that miRNA expression patterns will be useful as clinical markers to be correlated with parameters like disease prognosis, therapy related toxicity, and the likelihood to develop secondary malignancies.

Cloning efforts and bioinformatic predictions suggest that miRNAs may regulate up to 20%–25% of mammalian genes. The accurate profiling of miRNA expression thus represents an important tool to investigate physiological and pathophysiological states. Both the qualitative and the quantitative expressions of miRNAs, therefore, are expected to exert a profound regulatory influence on the transcriptome of a given cell or tissue

4. What led you to try out LNA™-based arrays for microRNA profiling?

Well, first of all we didn’t have much success with capture probes of conventional chemistry. We realized that, in contrast to our experience with mRNA expression profiling DNA-microarrays, we were not able to improve our results by playing with hybridization conditions, probe design or slide coating. We then hypothesized that LNA -modified capture probes: (1) may result in a more sensitive detection of miRNAs in comparison to unmodified DNA-based capture probes and (2) can be designed such that an uniform melting temperature (Tm) can be applied to a genome-wide set of miRNAs by adjusting the LNA content and the length of the capture probes. Tm normalization of capture probes permits the establishment of normalized hybridization conditions suitable for all miRNAs, which otherwise cover a range of Tm's between 45°C and 74°C.

5. In your opinion, what are the key differences between LNA™-based and DNA-based arrays when it comes to profiling microRNAs?

The main difference is selectivity and sensitivity. The biophysical properties of LNA allow the design of probe sets for uniform, high-affinity hybridizations. This yields highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. The way we established our experimental protocols will greatly simplify miRNA expression profiling of biological and clinical samples. At the end of the day selectivity and sensitivity means faster processing of biological samples and more answers than questions when it comes to data analysis and interpretation.

6. Currently, what is the biggest challenge to your miRNA research?

Technically, we are able to produce miRNA expression profiles and deduce meaningful fingerprints. Although such fingerprints will be extremely useful for clinical diagnostics, they tell us little about the mechanism of pathogenesis. The next challenge is to identify bona fide target genes and understand the regulatory networks associated with the expression of miRNAs.

7. Finally, can you give us an indication of your near-future research plans?

Currently, we are intensively working on studying miRNA dependent responses in two of our major research projects: iron metabolism and childhood leukemias.

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