Screening of Suitable Reference Genes for Immune Gene Expression Analysis Stimulated by Vibrio anguillarum and Copper Ions in Chinese Mitten Crab (Eriocheir sinensis)

The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we investigated gene selection, and the most stable reference gene for the Chinese mitten crab (Eriocheir sinensis) was screened under the stimulation of Vibrio anguillarum and copper ions, respectively. Ten candidate reference genes were selected, including arginine kinase (AK), ubiquitin-conjugating enzyme E2b (UBE), glutathione S-transferase (GST), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF-1α), α-tubulin (α-TUB), heat shock protein 90 (HSP90), β-actin (β-ACTIN), elongation factor 2 (EF-2) and phosphoglucomutase 2 (PGM2). Expression levels of these reference genes were detected under the stimulation of V. anguillarum at different times (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and copper ions in different concentrations (11.08 mg/L, 2.77 mg/L, 0.69 mg/L and 0.17 mg/L). Four types of analytical software, namely geNorm, BestKeeper, NormFinder and Ref-Finder, were applied to evaluate the reference gene stability. The results showed that the stability of the 10 candidate reference genes was in the following order: AK > EF-1α > α-TUB > GAPDH > UBE > β-ACTIN > EF-2 > PGM2 > GST > HSP90 under V. anguillarum stimulation. It was GAPDH > β-ACTIN > α-TUB > PGM2 > EF-1α > EF-2 > AK > GST > UBE > HSP90 under copper ion stimulation. The expression of E. sinensis Peroxiredoxin4 (EsPrx4) was detected when the most stable and least stable internal reference genes were selected, respectively. The results showed that reference genes with different stability had great influence on the accurate results of the target gene expression. In the Chinese mitten crab (E. sinensis), AK and EF-1α were the most suitable reference genes under the stimulation of V. anguillarum. Under the stimulation of copper ions, GAPDH and β-ACTIN were the most suitable reference genes. This study provided important information for further research on immune genes in V. anguillarum or copper ion stimulation.