Computational protocol: Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

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[…] Total, 10 commonly used reference genes, ACT3, ELF1A, G6PD, PEPKR1, TUB, TUA, CYP, GAPDH, H3, and 18S, were identified from the published literature. The corresponding gene sequences were cloned and sequenced based on kenaf transcriptome sequencing data (Chen et al., ), and the corresponding gene sequences has been submitted to NCBI ( On the basis of the corresponding gene sequences, primers were designed for RT-qPCR using the Primer 3 Plus program ( The primers of corresponding gene were tested using Primer Select program within the Lasergene 9 software (Burland, ) for dimerization and hairpin formation. [...] Four common statistical packages, GeNorm (Vandesompele et al., ), NormFinder (Andersen et al., ), ΔCt (Silver et al., ), and BestKeeper (Pfaffl et al., ), were implemented to estimate the stability of each candidate reference gene.For GeNorm and NormFinder, the Ct-values were transformed into relative quantities using the formula 2−ΔCt; ΔCt is the corresponding Ct-value minus the minimum Ct-value (Chen et al., ). GeNorm was used to analyze gene stability (M-value) and pairwise variation (Vn/n+1) value by pairwise comparisons among all of the reference genes. The M-value correlated with the expression stability, and the lowest M-values were considered the most stable. The threshold of 0.15 was the criterion value of Vn/n+1. A value less than 0.15 are not necessary to use a reference gene for the normalization of the RT-qPCR analysis (Vandesompele et al., ). The NormFinder approach is used to rank the candidate reference genes by estimating intra- and inter-group gene expression variations (Andersen et al., ). The reference gene with the lowest value was also considered to be the most stable. The ΔCt approach, which compares standard deviation (SD) values of each gene, was used to rank the candidate reference genes (Silver et al., ). BestKeeper ranked the candidate reference genes using the SD and the coefficients of variance (CVs), and the lowest SD value indicated the most stable gene. Based on the ranking from the four statistical approaches, RefFinder ( assigned an appropriate weight to each gene and calculated the geometric mean of their weights for the overall final ranking (Kim et al., ). [...] The combination of TUB, CYP, and PEPKR1, which was recommend by the RefFinder and GeNorm methods across all sample sets, was used as an internal control to differentiate the expressions of five mitochondria F1F0-ATPase subunit genes (atp1, atp4, atp6, atp8, and atp9) in lines 722A and 722B. The expression patterns of these genes were calculated using 2−ΔΔCt method. Least significant differences were determined by Duncan's test (P ≤ 0.05) using SPSS Statistics 21.0 (IBM). […]

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