Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns

Abstract

BackgroundResearch using the Pacific oyster Crassostrea gigas as a model organism has experienced rapid growth in recent years due to the development of high-throughput molecular technologies. As many as 56,268 EST sequences have been sequenced to date, representing a genome-wide resource that can be used for transcriptomic investigations.ResultsIn this paper, we developed a Pacific oyster microarray containing oligonucleotides representing 31,918 transcribed sequences selected from the publicly accessible GigasDatabase. This newly designed microarray was used to study the transcriptome of male and female gonads, mantle, gills, posterior adductor muscle, visceral ganglia, hemocytes, labial palps and digestive gland. Statistical analyses identified genes differentially expressed among tissues and clusters of tissue-enriched genes. These genes reflect major tissue-specific functions at the molecular level, such as tissue formation in the mantle, filtering in the gills and labial palps, and reproduction in the gonads. Hierarchical clustering predicted the involvement of unannotated genes in specific functional pathways such as the insulin/NPY pathway, an important pathway under study in our model species. Microarray data also accurately identified reference genes whose mRNA level appeared stable across all the analyzed tissues. Adp-ribosylation factor 1 (arf1) appeared to be the most robust reference for normalizing gene expression data across different tissues and is therefore proposed as a relevant reference gene for further gene expression analysis in the Pacific oyster.ConclusionsThis study provides a new transcriptomic tool for studies of oyster biology, which will help in the annotation of its genome and which identifies candidate reference genes for gene expression analysis.

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster

“…Among these three selected reference genes, arf1 encodes a conserved guanine nucleotide-binding protein that has a central role in intra-Golgi transport (Serafini et al, 1991;Li et al, 2004). Although it has been used as reference gene in human (Eisenberg and Levanon, 2003), arf1 of C. gigas was only revealed to be stably expressed in adult tissues in a recent microarray study (Dheilly et al, 2011). gapdh encodes a key enzyme in glucose metabolism and is one of the most commonly used reference genes in quantitative gene expression analysis (Huggett et al, 2005).…”

“…Selection of candidate housekeeping genes We collected 13 housekeeping genes as candidate internal reference genes (Table 1), including the three commonly used genes elf1α, gapdh and actin, and the arf1 gene, which was reported to be stably expressed in adult oyster tissues (Dheilly et al, 2011). The other nine genes were proposed as candidate reference genes by Du et al (2013).…”

“…Despite the increasing number of reports, however, studies on molluscan development are still in their infancy, especially when compared to model organisms such as the fruit fly and nematode. In recent years, the Pacific oyster Crassostrea gigas has emerged as a model mollusk species with the publication of the whole genome sequence (Zhang et al, 2012) and of fundamental studies (Fabioux et al, 2004;Badariotti et al, 2007;Dheilly et al, 2011;Huan et al, 2013). In most of these studies, quantitative gene expression analysis (mainly based on real-time PCR assay) is an important element, providing quantitative gene expression data to study gene function.…”

“…To date, two studies have assessed the expression stability of housekeeping genes in C. gigas. In 2011, a microarray analysis provided a list of housekeeping genes that are stably expressed in adult tissues and suggested that the adp-ribosylation factor 1 (arf1) gene should be used as the internal reference (Dheilly et al, 2011). More recently, Du et al (2013) conducted an expression analysis of housekeeping genes in larvae of C. gigas and suggested that two ribosomal protein genes be used for normalization.…”

Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

“…This platform has been used by scientists to study the expression profile in many species from yeast to human [24,25]. Microarrays have also been applied to bivalves for different purposes [26][27][28]. To our knowledge, a toxicogenomics study in the gill and the digestive gland [20] and a recent study of the response to brown ring disease, [29] are the only works to date to use an oligo-microarray in R. philippinarum.…”