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

The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genome-wide studies, accurate analyses of quantitative gene expression during development are impeded by the lack of validated reference genes. To improve the situation, in this study, we analyzed the expression stability of seven candidate housekeeping genes during early development of the Pacific oyster Crassostrea gigas: actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), α subunit of elongation factor 1 (elf1α), adp-ribosylation factor 1 (arf1), heterogeneous nuclear ribonucleoprotein q, ubiquitin-conjugating enzyme e2d2 and ribosomal protein s18. We focused on 11 stages from oocyte to D-veliger, which include crucial developmental processes such as axis determination, gastrulation and shell formation. Gene expression stabilities were assessed with the three commonly used programs geNorm, NormFinder and BestKeeper. Although the results obtained with the three programs varied to some extent, in general, arf1, elf1α and gapdh were highly ranked and actin was poorly ranked. This analysis also indicated that multiple genes should be used for normalization, and we concluded that arf1-elf1α-gapdh should be used as internal references. The findings of this study will help researchers to obtain accurate results in future quantitative gene expression analysis of development in bivalve mollusks.