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Logo of bmcgenoBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Genomics
 
BMC Genomics. 2009; 10: 195.
Published online Apr 27, 2009. doi:  10.1186/1471-2164-10-195
PMCID: PMC2684549
Parallel DNA pyrosequencing unveils new zebrafish microRNAs
Ana R Soares,1,2 Patrícia M Pereira,1 Bruno Santos,3 Conceição Egas,2,3 Ana C Gomes,3 Joel Arrais,4 José L Oliveira,4 Gabriela R Moura,1 and Manuel AS Santoscorresponding author1
1RNA Biology Laboratory, Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
2Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal
3Biocant Research Centre, 3060-197 Cantanhede, Portugal
4IEETA, University of Aveiro, 3810-193 Aveiro, Portugal
corresponding authorCorresponding author.
Ana R Soares: arsoares/at/cnc.cj.uc.pt; Patrícia M Pereira: pereirap/at/ua.pt; Bruno Santos: bsantos/at/biocant.pt; Conceição Egas: cegas/at/biocant.pt; Ana C Gomes: acgomes/at/biocant.pt; Joel Arrais: jpa/at/ua.pt; José L Oliveira: jlo/at/ua.pt; Gabriela R Moura: gmoura/at/ua.pt; Manuel AS Santos: msantos/at/ua.pt
Received November 4, 2008; Accepted April 27, 2009.
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.
Conclusion
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.
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