Identification of small non-coding RNAs in zebrafish

MicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification; however, cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes. We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from small non-coding RNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics and 25 novel miRNAs were predicted. Overall design: Small RNA libraries were prepared from different zebrafish developmental stages, namely, 24 hours post-fertilization (hpf), 72 hpf, 96 hpf, 5 days post-fertilization (dpf), 45 dpf, young adult and from adult brain, eyes, gills, heart, skin and fins.