Identification of valid reference genes for the normalization of RT-qPCR gene expression data in Alexandrium catenella under different nutritional conditions

Alexandrium catenella is a cosmopolitan harmful algal bloom (HAB) forming dinoflagellate. To comprehend its mechanisms of bloom formation, gene expression analysis is indispensable. Quantitative real-time reverse transcription PCR (qRT-PCR) is an ideal method for swift and precise quantification of gene expression analysis that greatly relies on selection of apposite reference genes for data normalization. To date, limited studies have focused on the screening of reference genes in dinoflagellates. The unavailability of valid reference gene for normalizing poses hindrances in the appliance of qRT-PCR to the HAB forming group. The work presented here evaluated 12 reference genes for their expression stability using qRT-PCR under diverse nutritional conditions together with high light. Statistical algorithm such as RefFinder was used that analyze data using geNorm and NormFinder, comparative delta-CT method along with a combination approach that declares comprehensive ranking contingent upon the geometric mean of the results procured from other methods. Comprehensive analysis across all condition by geNorm showed ACT, IPP, and GAPDH as genes possessing highest stability followed by Tubα and ICDH. Comprehensive analysis by Normfinder declared that IPP is the most stable gene followed by ACT, CYC, GAPDH, and GTEF. Combination approach using comparative delta CT, geNorm and NormFinder analysis programs through RefFinder as well as ranking by standard deviation of delta CT declared IPP as the most stable gene followed by ACT and GAPDH. IPP, ACT, and GAPDH represented the top 4 listed stable reference genes among all the analysis methods used. The results of pairwise variation by geNorm showed that V2/V3 value under all the tested conditions was below the cut-off value of 0.15 which shows that two genes are sufficient for normalization. Our results in accord with other widely conducted studies emphasize the significance of reference gene validation in precise experimental setup before appliance to avoid serious misinterpretation of the results. It is highly expected that the procedures and outcome of the report may be helpful for future studies in the gene expression analysis of A. catenella and to screen out reference genes for other algae.