Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system

Aims To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system. Methods and results Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR. Conclusions CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions. Significance and impact of the study The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks.