Selection and evaluation of stable reference genes for quantitative real-time PCR in the head kidney leukocyte of Oreochromis niloticus

To investigate the antibacterial mechanisms of Nile tilapia, it is necessary to understand the responses of immune-related genes in infected lymphocytes. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is applied to investigate transcriptional profiling. However, few studies have evaluated stable reference genes for qRT-PCR in Nile tilapia head kidney lymphocytes (HKLs). In this research, we assessed the stability of seven common reference genes, which include 18 S ribosomal RNA (18 s rRNA), beta actin (β-actin), beta-2-microglobulin-like (β-MG), elongation factor 1a (EF1A), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Tubulin alpha chain-like (TUBA), and Ubiquitin-conjugating enzyme (UBE)，in both resting state and several responding states of Nile tilapia HKLs. Results showed that β-MG was the optimum reference gene in both resting state and LTA stimulation state, while GAPDH was the optimum for LPS stimulation. However, no single candidate gene could be employed as the universal reference under all situations. Next, the geNorm and RefFinder tools was applied to access the adequacy of the union of two reference genes, and the results suggested that the combination of β-MG and GAPDH was the best candidate. Finally, the expression profiles of three typical immune-related genes interleukin-1β (IL-1β), transcription factor p65(P65), and tumor necrosis factor α (TNFα) under LPS/LTA situations in HKLs were evaluated via multiple reference genes and single reference gene difference analysis. The findings revealed that TUBA and UBE were incapable of working as reference genes, and the union of two reference genes was better than any sole reference gene. The single-cell RNA-Seq (scRNA-Seq) data of Nile tilapia HKLs confirmed the above results. In conclusion, our data suggest that the combination of β-MG and GAPDH may be the best criterion for qRT-PCR calculation in tilapia HKLs. •GAPDH and β-MG were the most stable reference genes for Nile tilapia HKLs in response to different stimuli.•Using two reference genes (β-MG and GAPDH) for normalization will be more suitable for Nile tilapia HKLs.•TUBE and UBE were not suitable as reference genes for tilapia HKLs.•β-actin, β-MG, EF1A and GAPDH were highly expressed and evenly distributed among different subgroups of HKLs.