Reference genes for gene expression normalization in Chlamydomonas sp ICE-L by quantitative real-time RT-PCR

The determination of a robust reference gene has become increasingly important since RT-qPCR used as a prominent technique for quantification of transcript in connection with their molecular and biological mechanisms. Only a few studies on reference genes have been conducted using Antarctic ice algae. In this work, 10 candidate reference genes of Chlamydomonas sp. ICE-L were evaluated for their stabilities. The results showed that the best references genes differed across the experimental samples. Based on NormFinder Analysis, EF-1 alpha was the most suitable reference gene under the diurnal cycle, high light, high salinity and UV-B irradiation conditions, and GAPDH was the most stable gene under different light intensities. For all tested samples H2B was the best gene and 18S was the least. Pair-wise variation analysis revealed that H2B and EF-1 alpha were the best gene combination for diurnal cycle and high light conditions. For different light intensities and high salinity samples, the best combinations were GAPDH + ACT and L32 + H2B, respectively. For UV-B irradiated samples, a minimum of three genes (EF-1 alpha, L32 and 18S) were necessary for accurate normalization. Selecting appropriate reference gene was very important to achieve an accurate and reliable normalization of genes' expression. These results provided guidelines for reference genes selection under different experimental conditions and also established a foundation for more accurate and widespread use of RT-qPCR in Chlamydomonas sp. ICE-L.