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Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna

Abstract

Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels. Accurate and reproducible results are dependent on the correct choice of reference genes for data normalization. Atropa belladonna is a commercial plant species from which pharmaceutical tropane alkaloids are extracted. In this study, eight candidate reference genes, namely 18S ribosomal RNA (18S), actin (ACT), cyclophilin (CYC), elongation factor 1α (EF-1α), β-fructosidase (FRU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and beta-tubulin (TUB), were selected and their expression stabilities studied to determine their suitability for normalizing gene expression in A. belladonna. The expression stabilities of these genes were analyzed in the root, stem, and leaf under cold, heat, NaCl, UV-B, methyl jasmonate, salicylic acid, and abscisic acid treatments using geNorm, NormFinder, and BestKeeper. The statistical algorithms indicated that PGK was a reliable gene for normalizing gene expression under most of the experimental conditions. The pairwise value analysis showed that two genes were sufficient for proper expression normalization, except when analyzing gene expression in heat-treated roots. However, the choice of the second reference gene depended on specific conditions. Finally, the relative expression level of the PMT gene of A. belladonna was detected to validate the selection of PGK a reliable reference gene. In summary, our results should guide the selection of appropriate reference genes for gene expression studies in A. belladonna under different organs and abiotic stress conditions.

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Acknowledgments

This work was supported financially by the NSFC project (31370333), the Program for New Century Excellent Talents in University (NCET-12-0930), the National 863 Hi-Tech Plans (2011AA100605; 2011AA100607), Chongqing Sciences and Technology Project (CSTC2012GGYYJS80013), and the Fundamental Research Funds for the Central Universities (XDJK2013A024).

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Correspondence to Zhihua Liao or Kexuan Tang.

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Li, J., Chen, M., Qiu, F. et al. Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna . Plant Mol Biol Rep 32, 1002–1014 (2014). https://doi.org/10.1007/s11105-014-0701-9

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  • DOI: https://doi.org/10.1007/s11105-014-0701-9

Keywords

  • Atropa belladonna
  • Gene expression
  • Normalization
  • qPCR
  • Reference gene