Profiling bovine blastocyst microRNAs using deep sequencing.
MicroRNAs (miRNAs) are known to control several reproductive functions, including oocyte maturation, implantation and early embryonic development. Recent advances in deep sequencing have allowed the analysis of all miRNAs of a sample. However, when working with embryos, due to the low RNA content, miRNA profiling is challenging because of the relatively large amount of total RNA required for library preparation protocols. In the present study we compared three different procedures for RNA extraction and prepared libraries using pools of 30 bovine blastocysts. In total, 14 of the 15 most abundantly expressed miRNAs were common to all three procedures. Furthermore, using miRDeep discovery and annotation software (Max Delbrück Center), we identified 1363 miRNA sequences, of which bta-miR-10b and bta-miR-378 were the most abundant. Most of the 179 genes identified as experimentally validated (86.6%) or predicted targets (13.4%) were associated with cancer canonical pathways. We conclude that reliable analysis of bovine blastocyst miRNAs can be achieved using the procedures described herein. The repeatability of the results across different procedures and independent replicates, as well as their consistency with results obtained in other species, support the biological relevance of these miRNAs and of the gene pathways they modulate in early embryogenesis.
Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territori, Università degli Studi di Milano, Via Celoria 2, 20133, Milan, Italy.
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Pasquariello
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Dublin, Ireland.
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B
Fernandez-Fuertes
Parco Tecnologico Padano, Via Einstein Albert, 26900, Lodi, Italy.
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F
Strozzi
Istituto di Biologia e Biotecnologia Agraria - Consiglio Nazionale delle Ricerche, Via Einstein Albert, 26900, Lodi, Italy.
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Pizzi
Associazione Italiana Allevatori, Via Bergamo 292, 26100, Cremona, Italy.
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Mazza
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Dublin, Ireland.
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P
Lonergan
Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territori, Università degli Studi di Milano, Via Celoria 2, 20133, Milan, Italy.
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Gandolfi
School of Animal and Veterinary Sciences, Faculty of Science, University of Adelaide, Roseworthy, SA 5371, Australia.
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Williams
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle; D044127:Epigenesis, Genetic; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D059014:High-Throughput Nucleotide Sequencing; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy
D035683:MicroRNAs