The accurate measurement of gene expression via quantitative real-time reverse transcription PCR(q RT-PCR)heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA(18S), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), α-tubulin(TUA), β-tubulin(TUB), actin(ACT) and cytochrome oxidase subunit 1(COX1), were evaluated for their expression stability with q RT-PCR and three statistical algorithms(Ge Norm, Norm Finder, and Best Keeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages(resting cysts), different growth stages, in darkness, exposed to abscisic acid(ABA) and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression a
The accurate measurement of gene expression via quantitative real-time reverse transcription PCR(q RT-PCR)heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA(18S), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), α-tubulin(TUA), β-tubulin(TUB), actin(ACT) and cytochrome oxidase subunit 1(COX1), were evaluated for their expression stability with q RT-PCR and three statistical algorithms(Ge Norm, Norm Finder, and Best Keeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages(resting cysts), different growth stages, in darkness, exposed to abscisic acid(ABA) and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates r