Discovering miRNAs: Ph.D. thesis

MicroRNAs (miRNAs) are a class of non-coding RNAs that act as negative regulators of gene expression. Here I present the identification of six new miRNAs. Furthermore, the involvement of miRNAs in osteoblast differentiation is investigated. Artificial regulation of miR-34a, miR-92, and miR-210 is shown to affect osteoblast differentiation of mesenchymal stem cells (MSCs). However, no miRNAs showing an expression pattern dependent on this differentiation was found. In stead four miRNAs (miR-7, miR-26b, miR-34a, and miR-210) are identified which expression pattern shows dependency on cell density. This cell density dependent expression is found to be conserved in several human cell lines. I also present data indicating that miR-17-5p and miR-20a are involved in both proper spermatogenesis as well as development of testis cancer. I played a large role in implementation of the microarray technique in our laboratory. I also developed an experimental approach for identification of miRNA targets capable of selecting mRNAs showing complementarity towards a specific miRNA. In a test of LNA knockdown probes I found that probes based on deoxyribose nucleotides or on 2’-O-methyl modified nucleotides works better than probes based on ribose nucleotides. Finally, a miRNA detection method relying on padlock probes is presented. This method is shown to discriminate between closely related miRNAs and only require few nanograms of total RNA for detection.