RT-qPCR normalization of reference genes in different life-history stages of Gracilaria vermiculophylla (Rhodophyta)

The quantitative realtime polymerase chain reaction (RT-qPCR) is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization. We evaluated the expressions of 8 housekeeping genes: 18S ribosomal rDNA ( 18S rDNA ), 28S ribosomal rDNA ( 28S rDNA ), rubisco large subunit ( rbc-L ), β-actin ( ACT ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), elongation factor 1 ( EF1 ), β-tubulin ( TubB ), and P-phycoerythrin B ( PEB ), to select the suitable reference genes for different life-history stages (tetrasporophyte, carposporophyte, and male/female gametophyte) of Gracilaria vermiculophylla by absolute quantitative method. Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis. Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages. It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases. 18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples. But for samples with different maturities, TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.