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Open Access Highly Accessed Research article

RNA-seq analysis of single bovine blastocysts

James L Chitwood1, Gonzalo Rincon1, German G Kaiser2, Juan F Medrano1 and Pablo J Ross1*

Author Affiliations

1 Department of Animal Science, University of California, Davis, 1 Shields Avenue, Davis, CA, USA

2 Laboratorio de Biotechnología de la Reproducción, INTA, Balcarce, Argentina

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BMC Genomics 2013, 14:350  doi:10.1186/1471-2164-14-350

Published: 25 May 2013

Abstract

Background

Use of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect.

Results

We report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant’s transcripts.

Conclusions

This study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression.

Keywords:
RNA-seq; Bovine; Embryo; Blastocyst; SNP; ASE