Validation of housekeeping genes for quantitative mRNA expression analysis in OsHV-1 infected ark clam, Scapharca broughtonii.

Ostreid herpesvirus-1 (OsHV-1) presents interspecies transmission among bivalves. Recently, events of mass mortalities of ark clams (Scapharca broughtonii) infected with OsHV-1 have been recorded. To accurately assess the gene responding patterns of ark clams post OsHV-1 infection, constant stable housekeeping genes (HKGs) are needed as internal control to normalize raw mRNA expression data. In this study, ten candidate HKGs were selected, including 18S rRNA (18S), beta-actin (ACT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase subunit (NADH), Elongation factor-1a (EF-1a), Elongation factor-1ß (EF-1ß), Elongation factor-1γ (EF-1γ), Ribosomal protein L7 (RL7), Ribosomal protein L15 (RL15) and Ribosomal protein S18 (S18). The expression levels of ten candidate HKGs were analyzed by real-time PCR under given experimental conditions, including various tissues, OsHV-1 challenge, temperature stress and OsHV-1 challenge at different temperature. Their expression stability values were further calculated using two different statistical models (geNorm and NormFinder). The results showed that different tissues presented distinct best pair genes combinations for gene expression analysis under OsHV-1 challenge. RL15 was comparatively more stable than other HKGs under various experimental conditions, while commonly used 18s and ACT seemed to be more greatly influenced by most given experimental conditions in ark clams. This study emphasized the necessity of prior validation of HKGs and would facilitate future gene expression analysis in ark clams or other shellfishes.