Abstract
RNA sequencing (RNA-Seq)-based gene expression analysis is applicable to a wide range of biological purposes in various species. Reverse transcription quantitative PCR (RT-qPCR) is also used to assess target gene expression utilizing stably expressed reference genes as internal control under a given set of conditions. However, investigations of the reference genes for RT-qPCR normalization in the process of somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum are rarely reported. In this study, on the basis of our previous transcriptome data of three different induction stages during SE initial dedifferentiation process in four G. hirsutum cultivars that have different SE capability, 15 candidate genes were selected during SE initial dedifferentiation process, and their expression stability was evaluated by geNorm, NormFinder, and BestKeeper. The results indicated that the two genes of endonuclease 4 (ENDO4) and 18S ribosomal RNA (18S rRNA) showed stable expression in the four different G. hirsutum cultivars, endowing them to be appropriate reference genes during three induction stages in the four cotton cultivars. In addition, the stability and reliability of the two reference genes of ENDO4 and 18S rRNA were further verified by comparing the expressions of auxin-responsive protein 22 (AUX22) and ethylene-responsive transcription factor 17 (ERF17) between RT-qPCR results and the RNA-seq data, which showed strong positive correlation coefficient (R2 = 0.8396–0.9984), validating again the steady expression of ENDO4 and 18S rRNA as the reliable reference genes. Our results provide effective reference genes for RT-qPCR normalization during SE process in different G. hirsutum cultivars.
Highlights
Gene expression level analysis is crucial in many fields of biological research [1]
17 (ERF17) and auxin-responsive protein 22 (AUX22) with altered expressions were further selected to validate the expression stability of endonuclease 4 (ENDO4) and 18S rRNA, showing again the highly steady expression of the two genes. All these results suggest that ENDO4 and 18S rRNA can be used as appropriate reference genes for Reverse transcription quantitative PCR (RT-qPCR) normalization during somatic embryogenesis (SE) initial dedifferentiation in different G. hirsutum cultivars
According to our previous RNA sequencing (RNA-Seq)-based transcriptome data of three different induction stages during SE initial dedifferentiation process in four G. hirsutum cultivars that have different SE capability, the screen rules of q-value ≥ 0.05, FPKM ≥ 5, |log2FoldChange| < 1, and relatively lower coefficient of variance (CV) of FPKM were set as the higher criteria at all sampling points to select the candidate reference genes
Summary
Gene expression level analysis is crucial in many fields of biological research [1]. RNA sequencing (RNA-Seq) has become the prevalent method used to analyze the transcriptional gene expression levels in various species [2,3]. Reverse transcription quantitative PCR (RT-qPCR) is the widely applied method to quantify gene expression and to validate transcriptomic data [4,5] for its prominent advantages of high sensitivity, reproducibility, and specificity [6]. Inaccurate quantification and low-quality RNA may lead to a decrease in RT-qPCR specificity [7]. It is necessary and crucial to screen stably expressed reference genes to normalize target gene expression level under a given set of conditions. Reliable reference genes are usually selected from the stably
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