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Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions

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Abstract

Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr6+) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica.

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References

  • Artico S, Nardeli SM, Brilhante O, Grossi-de-Sa MF, Alves-Ferreira M (2010) Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol 10:49

    Article  PubMed  PubMed Central  Google Scholar 

  • Chandna R, Augustine R, Bisht NC (2012) Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One 7(5):e36918

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Chen H, Lai Z, Shi J, Xiao Y, Chen Z, Xu X (2010a) Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biol 10:281

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Chen X, Truksa M, Shah S, Weselake RJ (2010b) A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus. Anal Biochem 405(1):138–140

    Article  PubMed  CAS  Google Scholar 

  • Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X (2011) BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol 11:136

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139(1):5–17

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • de Carvalho K, Bespalhok Filho JC, dos Santos TB, de Souza SG, Vieira LG, Pereira LF, Domingues DS (2013) Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): identification and validation of new genes for qPCR normalization. Mol Biotechnol 53(3):315–325

    Article  PubMed  Google Scholar 

  • Demidenko NV, Logacheva MD, Penin AA (2011) Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data. PLoS One 6(5):e19434

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Fang X, Willis RC, Hoang Q, Kelnar K, Xu W (2004) High-throughput sample preparation for gene expression profiling and in vitro target validation. JALA 9(3):140–145

    CAS  Google Scholar 

  • Gamm M, Heloir MC, Kelloniemi J, Poinssot B, Wendehenne D, Adrian M (2011) Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis. Mol Genet Genomics 285(4):273–285

    Article  PubMed  CAS  Google Scholar 

  • Garg H, Sivasithamparam K, Banga S, Barbetti M (2008) Cotyledon assay as a rapid and reliable method of screening for resistance against Sclerotinia sclerotiorum in Brassica napus genotypes. Australas Plant Pathol 37(2):106–111

    Article  Google Scholar 

  • Goossens K, Van Poucke M, Van Soom A, Vandesompele J, Van Zeveren A, Peelman LJ (2005) Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev Biol 5:27

    Article  PubMed  PubMed Central  Google Scholar 

  • Gu C, Chen S, Liu Z, Shan H, Luo H, Guan Z, Chen F (2011) Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol 49(2):192–197

    Article  PubMed  CAS  Google Scholar 

  • Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60(2):487–493

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6(6):609–618

    Article  PubMed  CAS  Google Scholar 

  • Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112

    Article  PubMed  PubMed Central  Google Scholar 

  • Huis R, Hawkins S, Neutelings G (2010) Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol 10:71

    Article  PubMed  PubMed Central  Google Scholar 

  • Janska A, Hodek J, Svoboda P, Zamecnik J, Prasil IT, Vlasakova E, Milella L, Ovesna J (2013) The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress. Mol Genet Genomics 288:639–649

    Article  PubMed  CAS  Google Scholar 

  • Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59

    Article  PubMed  PubMed Central  Google Scholar 

  • Kim BR, Nam HY, Kim SU, Kim SI, Chang YJ (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25(21):1869–1872

    Article  PubMed  CAS  Google Scholar 

  • Klie M, Debener T (2011) Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida). BMC Res Notes 4:518

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Kundu A, Patel A, Pal A (2013) Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo. Plant Cell Rep 32(10):1647–1658

    Article  PubMed  CAS  Google Scholar 

  • Le DT, Aldrich DL, Valliyodan B, Watanabe Y, Ha CV, Nishiyama R, Guttikonda SK, Quach TN, Gutierrez-Gonzalez JJ, Tran LS, Nguyen HT (2012) Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS One 7(9):e46487

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Liu D, Shi L, Han C, Yu J, Li D, Zhang Y (2012) Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS One 7(9):e46451

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Mascia T, Santovito E, Gallitelli D, Cillo F (2010) Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Mol Plant Pathol 11(6):805–816

    Article  PubMed  CAS  Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56(421):2907–2914

    Article  PubMed  CAS  Google Scholar 

  • Obrero A, Die JV, Roman B, Gomez P, Nadal S, Gonzalez-Verdejo CI (2011) Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR. J Agric Food Chem 59(10):5402–5411

    Article  PubMed  CAS  Google Scholar 

  • Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:11

    Article  PubMed  PubMed Central  Google Scholar 

  • Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Method 46(1–2):69–81

    Article  CAS  Google Scholar 

  • Stajner N, Cregeen S, Javornik B (2013) Evaluation of reference genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with vascular pathogen Verticillium albo-atrum. PLoS One 8(7):e68228

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Sturzenbaum SR, Kille P (2001) Control genes in quantitative molecular biological techniques: the variability of invariance. Comp Biochem Phys B 130(3):281–289

    Article  CAS  Google Scholar 

  • Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C (2002) Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 309(2):293–300

    Article  PubMed  CAS  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):research0034

    Article  PubMed  PubMed Central  Google Scholar 

  • Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399(2):257–261

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Yu K, Poysa V, Shi C, Zhou Y (2012a) Selection of reference genes for normalization of qRT-PCR analysis of differentially expressed genes in soybean exposed to cadmium. Mol Biol Rep 39(2):1585–1594

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Tan X, Zhang Z, Gu S, Li G, Shi H (2012b) Defense to Sclerotinia sclerotiorum in oilseed rape is associated with the sequential activations of salicylic acid signaling and jasmonic acid signaling. Plant Sci 184:75–82

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Wang Y, Zhou P (2013) Validation of reference genes for quantitative real-time PCR during Chinese wolfberry fruit development. Plant Physiol Biochem 70:304–310

    Article  PubMed  CAS  Google Scholar 

  • Warzybok A, Migocka M (2013) Reliable reference genes for normalization of gene expression in cucumber grown under different nitrogen nutrition. PLoS One 8(9):e72887

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Xiao D, Zhang NW, Zhao JJ, Bonnema G, Hou XL (2012) Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage. Funct Plant Biol 39(4):342–350

    Article  CAS  Google Scholar 

  • Xu J, Xu Z, Zhu Y, Luo H, Qian J, Ji A, Hu Y, Sun W, Wang B, Song J, Sun C, Chen S (2013) Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum. Curr Microbiol 68(1):120–126

    Article  PubMed  Google Scholar 

  • Zhong HY, Chen JW, Li CQ, Chen L, Wu JY, Chen JY, Lu WJ, Li JG (2011) Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions. Plant Cell Rep 30(4):641–653

    Article  PubMed  CAS  Google Scholar 

  • Zhu J, Zhang L, Li W, Han S, Yang W, Qi L (2013) Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS One 8(1):e53196

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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Acknowledgments

This work was funded by the National Natural Science Foundation of China (Nos. 31071672, 31271760, and 31071030), the Natural Science Foundation of Jiangsu Province (BK2010328), the China Postdoctoral Science Foundation Funded Project (2011M500873 and 2013T60507), the Natural Science Fund for Colleges and Universities in Jiangsu Province of China (10KJB210001), the Jiangsu Province Postdoctoral Science Foundation Funded Project (1102130C), and Jiangsu University (09JDG061).

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Authors and Affiliations

  1. Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang, 212013, People’s Republic of China

    Zheng Wang, Yu Chen, Hedi Fang, Haifeng Shi, Keping Chen, Zhiyan Zhang & Xiaoli Tan

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  1. Zheng Wang
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  2. Yu Chen
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  3. Hedi Fang
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  4. Haifeng Shi
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  5. Keping Chen
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  6. Zhiyan Zhang
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  7. Xiaoli Tan
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Correspondence to Zheng Wang or Xiaoli Tan.

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Communicated by S. Hohmann.

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Wang, Z., Chen, Y., Fang, H. et al. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions. Mol Genet Genomics 289, 1023–1035 (2014). https://doi.org/10.1007/s00438-014-0853-1

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