β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus.

The housekeeping gene encoding β-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to β-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.