Validation of housekeeping genes for gene expression studies in an ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> during freezing acclimation |
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Authors: | Chenlin Liu Guangting Wu Xiaohang Huang Shenghao Liu Bailin Cong |
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Institution: | (1) The First Institute of Oceanography, Marine Ecological Center, State Oceanic Administration, Qingdao, 266061, China |
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Abstract: | Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular
acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference
genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most
and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental
conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we
propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to
normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate
category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze
acclimation in Chlamydomonas sp. ICE-L. |
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