title: Parallel DNA pyrosequencing unveils new zebrafish microRNAs.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from miRNAs 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. Twenty five novel miRNAs were predicted, 107 miRNA star sequences and also 41 candidate miRNA targets were identified. A miRNA expression profile built on the basis of pyrosequencing read numbers showed high expression of most miRNAs throughout zebrafish development and identified tissue specific miRNAs.
  
  
  CONCLUSIONS
  This study increases the number of zebrafish miRNAs from 192 to 217 and demonstrates that a single DNA mini-chip pyrosequencing run is effective in miRNA identification in zebrafish. This methodology also produced sufficient information to elucidate miRNA expression patterns during development and in differentiated organs. Moreover, some zebrafish miRNA star sequences were more abundant than their corresponding miRNAs, suggesting a functional role for the former in gene expression control in this vertebrate model organism.
authors.affiliation: RNA Biology Laboratory, Department of Biology & CESAM, University of Aveiro, Aveiro 3810-193, Portugal. arsoares@cnc.cj.uc.pt
authors.forename: Ana R
authors.initials: AR
authors.lastname: Soares
authors.forename: Patrícia M
authors.initials: PM
authors.lastname: Pereira
authors.forename: Bruno
authors.initials: B
authors.lastname: Santos
authors.forename: Conceição
authors.initials: C
authors.lastname: Egas
authors.forename: Ana C
authors.initials: AC
authors.lastname: Gomes
authors.forename: Joel
authors.initials: J
authors.lastname: Arrais
authors.forename: José L
authors.initials: JL
authors.lastname: Oliveira
authors.forename: Gabriela R
authors.initials: GR
authors.lastname: Moura
authors.forename: Manuel A S
authors.initials: MA
authors.lastname: Santos
mesh_terms: D000818:Animals; D001483:Base Sequence; D019295:Computational Biology; D020869:Gene Expression Profiling; D015723:Gene Library; D008018:Life Cycle Stages / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017422:Sequence Analysis, DNA / Q000379:methods*; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development
chemical_list: D035683:MicroRNAs