MicroRNAs in the Spleen and Liver of the Developing Chick Embryo.

Abstract

MicroRNAs are small (˜19-24nt) non-coding RNAs that are involved in the regulation of gene expression. They are mainly expressed in development and many are expressed in a temporal as well as, a spatial manner. It is thought they may regulate up to 30% of all genes. Pyrosequencing using 454 Life Science technology is becoming the preferred method for microRNA profiling ad sequencing compared to the previous method of cloning and using traditional sequencing techniques. Use of 454 Life Sciences technology allows for a greater coverage of microRNAs and increases the chances of sequence low abundance microRNAs. In the current project we created four small RNA libraries from embryonic chick tissues, the spleen and liver at developmental time points E15 and E20. These libraries were then sequenced using 454 Life Sciences pyrosequencing. A total of 92,919 sequence reads were obtained, representing a total of 52,001 known chicken microRNAs. Of these 92,919 reads, 52,001 represented miRNAs matching the miRBase G. gallus database, and 3,472 were not found in the G. gallus database but were homologues of miRBase miRNAs from other species. Of these homologous reads 391 represented potential novel miRNAs. Other small RNAs, such as tRNA and rRNA, represented 24,672 of the reads, and 12,383 reads represented other types of sequences, such as degraded mRNA. More than one hundred different known miRNAs were identified in this study, and many were expressed in all four libraries. Common miRNAs that yielded multiple reads from all four libraries included miR-125b and miR-21, which are involved in general processes of cellular proliferation. Overall, the spleen libraries had a larger array of miRNAs than the liver libraries. Much of spleen development occurs during the later stages of embryonic development, so we can reasonably expect that many gene expression changes occur during these stages. As a result of this study, we identified nine potential novel chicken miRNAs. These novel miRNAs appear to be tissue-specific. The potential novel miRNAs appeared to be expressed at lower levels than some of the known miRNAs, which could indicate that most of the highly-expressed chicken miRNAs have already been identified, whereas, for the most part, the miRNAs expressed at low levels remain to be discovered.