Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development.
Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares--geNorm, NormFinder and BestKeeper--were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1α (elongation factor 1-α) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1β (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China.
Xiaojiao
X
Han
Mengzhu
M
Lu
Yicun
Y
Chen
Zhiyong
Z
Zhan
Qinqin
Q
Cui
Yangdong
Y
Wang
D000199:Actins / Q000235:genetics; D031284:Aleurites / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling; D020043:Genes, Essential / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics*; D025801:Ubiquitin / Q000235:genetics
D000199:Actins; D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D025801:Ubiquitin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases