Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.
Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
Shaona
S
Cao
Xiaowen
X
Zhang
Naihao
N
Ye
Xiao
X
Fan
Shanli
S
Mou
Dong
D
Xu
Chengwei
C
Liang
Yitao
Y
Wang
Wenqi
W
Wang
D000199:Actins / Q000235:genetics; D020418:Algal Proteins; D021983:Cyclophilins / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000235:genetics; D020871:RNA Stability; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D058009:Stramenopiles / Q000235:genetics*; D014404:Tubulin / Q000235:genetics
D000199:Actins; D020418:Algal Proteins; D014404:Tubulin; D005992:Glycerol-3-Phosphate O-Acyltransferase; D021983:Cyclophilins