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

Abstract

The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genomewide 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.

Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail,Lymnaea stagnalis

“…However, TUBA was unstable in C. nemoralis (Affenzeller, Cerveau & Jackson, 2018) and we also found Lst-TUBB to be highly unstable. ACTB has generally produced negative results as a candidate reference gene in molluscs (Cubero-Leon et al, 2012; Moreira et al, 2014; Liu et al, 2015; García-Fernández et al, 2016; Huan, Wang & Liu, 2016) and the results here from L. stagnalis are largely congruent with these previous findings, although Lst-ACTB was stable specifically in the foot. Finally, Rapac1 and VGKC were identified as suitable reference genes in C. nemoralis (Affenzeller, Cerveau & Jackson, 2018).…”

“…In one study of L. stagnalis , EF1α was identified to remain stable in the CNS under heat stress (Foster, Lukowiak & Henry, 2015). Given that EF1 α has also been identified to be highly stable in several other molluscs, it appears to be a strong candidate reference gene across the phylum (Morga et al, 2010; Wan et al, 2011; Cubero-Leon et al, 2012; Mauriz et al, 2012; Moreira et al, 2014; García-Fernández et al, 2016; Huan, Wang & Liu, 2016). An analysis of reference genes in the freshwater snail Bellamya aeruginosa showed that EF1 α and GAPDH were stable in the tentacles and penis but more variable in the foot (Liu et al, 2015), similar to our findings (albeit with some discrepancies of rankings in the tentacles and foot).…”

Reference genes for accurate evaluation of expression levels in Trichophyton interdigitale grown under different carbon sources, pH levels and phosphate levels

“…The ADP ribosylation factor gene ( adp-rf ) was found to be the best reference gene for analyses of target transcript levels in an exotic invasive insect, Leptinotarsa decemlineata 32 ; the melon Cucumis melo L. 33 ; the cereal wheats Triticum spp. 12 , the Pacific oyster, Crassostrea gigas 34 ; the monkey Macaca fascicularis 35 ; and the desert willow shrub, Salix psammophila 36 . Again, our previous study 11 demonstrated that the expression stability of adp-rf was low in M. canis .…”

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury inOnchidium reevesii

“…Certain housekeeping genes, such as 18S ribosomal RNA ( 18SrRNA ), 28S ribosomal RNA ( 28SrRNA ), β-actin ( ACTB ), cyclophilin ( CYP2 ), elongation factor 1-alpha ( EF1a ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), translation elongation factor ( TEF ), tubulin ( TUBB) and polyubiquitin ( UBQ ) (Huan, Wang & Liu, 2017; Song et al, 2017; Gao et al, 2017) are commonly used as reference genes. As these housekeeping genes are related to basic metabolic processes and are essential for normal cell growth, their expression levels are thought to be stable.…”