Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR (qRT-PCR) has been widely used for gene expression analysis, and selection of reference genes is a key point to obtain accurate results. To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia, different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress (DO 0.5 mg L-1 and DO 2.0 mg L-1) and normal condition (DO 7.5 mg L-1). Seven candidate reference genes were selected to evaluate the stability of their expression levels. The reference genes were evaluated by Delta Ct, BestKeeper, NormFinder and geNorm, and then screened by RefFinder calculation. Under hypoxic stress of 0.5 mg L-1, the most suitable reference gene for gill and hepatopancreas was RPL31, and the optimal reference genes for axe foot and adductor muscle were TUB and HIS, respectively. For hypoxic stress of 2.0 mg L-1, the most stable reference gene for gill and hepatopancreas was RPL31, and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A, respectively. At the normal condition, HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively, and GFRP2 was the best internal reference gene for axe foot and adductor muscle. The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress, which might be helpful for the analysis of other molluscs too.