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. 2023 Oct 15;24(20):15198.
doi: 10.3390/ijms242015198.

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii

Chaofan Jiang  1   2   3 , Lin Zhou  1   2   3 , Qingchen Zhao  1   2   3 , Mengke Wang  1   2   3 , Sirui Shen  1   2   3 , Te Zhao  1   2   3 , Kaidi Cui  1   2   3 , Leiming He  1   2   3
Affiliations

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii

Chaofan Jiang et al. Int J Mol Sci. .

Abstract

Reference genes are important for the accuracy of gene expression profiles using reverse-transcription quantitative PCR (RT-qPCR). However, there are no available reference genes reported for Sclerotium rolfsii; it actually has a pretty diverse and wide host range. In this study, seven candidate reference genes (UBC, β-TUB, 28S, 18S, PGK, EF1α and GAPDH) were validated for their expression stability in S. rolfsii under conditions of different developmental stages, populations, fungicide treatments, photoperiods and pHs. Four algorithm programs (geNorm, Normfinder, Bestkeeper and ΔCt) were used to evaluate the gene expression stability, and RefFinder was used to integrate the ranking results of four programs. Two reference genes were recommended by RefFinder for RT-qPCR normalization in S. rolfsii. The suitable reference genes were GAPDH and UBC across developmental stages, PGK and UBC across populations, GAPDH and PGK across fungicide treatments, EF1α and PGK across photoperiods, β-TUB and EF1α across pHs and PGK and GAPDH across all samples. Four target genes (atrB, PacC, WC1 and CAT) were selected for the validation of the suitability of selected reference genes. However, using one or two reference genes in combination to normalize the expression of target genes showed no significant difference in S. rolfsii. In short, this study provided reliable reference genes for studying the expression and function of genes in S. rolfsii.

Keywords: RT-qPCR; Sclerotium rolfsii; expression stability; reference gene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression levels (Ct values) of the seven candidate reference genes in S. rolfsii under all experimental conditions. The bars represent the standard deviation.
Figure 2
Figure 2
The expression stability order of seven candidate reference genes in S. rolfsii according to RefFinder across conditions of (A) developmental stages (mycelia and sclerotia), (B) populations (three MCGs), (C) fungicides (tebuconazole, prothioconazole, thifluzamide, carboxin and azoxystrobin), (D) photoperiods (24 h of dark and light), (E) pHs (5, 7 and 9), and (F) all samples. For (B–E), mycelia were used.
Figure 3
Figure 3
Pairwise variation (V) analysis of seven candidate reference genes calculated by geNorm under different experimental conditions. A cut-off value of 0.15 was used to determine the optimal number of reference genes.
Figure 4
Figure 4
Relative expression of target genes in S. rolfsii at different experimental conditions. (A) Expression profiles of CAT at different developmental stages. (B) Expression profiles of WC1 under different photoperiods. (C) Expression profiles of PacC under different pHs. (D) Expression profiles of atrB under different fungicide treatments. The expression profiles of target genes were normalized by the two most stably expressed (single and in combination) and the least stably expressed reference genes recommend by RefFinder. Values are means ± SD (n = 3). Different letters on the bars indicate significant differences between different treatments (p < 0.05, Tukey’s HSD test).
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References

    1. Bustin S.A., Benes V., Nolan T., Pfaffl M.W. Quantitative real-time RT-PCR—A perspective. J. Mol. Endocrinol. 2005;34:597–601. doi: 10.1677/jme.1.01755. - DOI - PubMed
    1. Huggett J., Dheda K., Bustin S., Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005;6:279–284. doi: 10.1038/sj.gene.6364190. - DOI - PubMed
    1. Tang J., Liang G., Dong S., Shan S., Zhao M., Guo X. Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization in Athetis dissimilis (Lepidoptera: Noctuidae) Under Different Conditions. Front. Physiol. 2022;13:842195. doi: 10.3389/fphys.2022.842195. - DOI - PMC - PubMed
    1. Hao X., Horvath D.P., Chao W.S., Yang Y., Wang X., Xiao B. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze) Int. J. Mol. Sci. 2014;15:22155–22172. doi: 10.3390/ijms151222155. - DOI - PMC - PubMed
    1. Wang B., Duan H., Chong P., Su S., Shan L., Yi D., Wang L., Li Y. Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum. Sci. Rep. 2020;10:15891. doi: 10.1038/s41598-020-73059-3. - DOI - PMC - PubMed

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