Analysis of gene expression level by RNA sequencing (RNA-seq ) has a wide range of biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) evaluated gene expression levels and validated transcriptomic, which will depend on the stably expressed reference genes for normalization of the gene expression level under specific situations. In this study, 15 candidate genes were selected from transcriptome datasets during somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes under all conditions. The stability and reliability of the reference genes were further tested through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription factor (ERF17). In summary, the results of our study indicate the most suitable reference genes for qRT-PCR during three induction stages in four cotton species.
Analysis of gene expression level by RNA sequencing (RNA-seq) has a wide range of 8 biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) 9 evaluated gene expression levels and validated transcriptomic, which will depend on the stably 10 expressed reference genes for normalization of the gene expression level under specific 11 situations. In this study, 15 candidate genes were selected from transcriptome datasets during 12 somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE 13 capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were 14 used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes 15 under all conditions. The stability and reliability of the reference genes were further tested 16 through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the 17 expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription 18 factor (ERF17). In summary, the results of our study indicate the most suitable reference genes 19 for qRT-PCR during three induction stages in four cotton species. 20
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