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Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR

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Abstract

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys2/His2-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis.

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References

  • Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250

    Article  PubMed  CAS  Google Scholar 

  • Brand Y, Hovav R (2010) Identification of suitable internal control genes for quantitative real-time PCR expression analyses in peanut (Arachis hypogaea). Peanut Sci 37:12–19

    Article  Google Scholar 

  • Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14

    Article  PubMed  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocr 25:169–193

    Article  CAS  Google Scholar 

  • Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–29

    Article  PubMed  CAS  Google Scholar 

  • Charrier B, Champion A, Henry Y, Kreis M (2002) Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol 130:577–590

    Article  PubMed  CAS  Google Scholar 

  • Chen ZB, Wang ML, Barkley NA, Pittman RN (2010) A simple allele-specific PCR assay for detecting FAD2 alleles in both A and B genomes of the cultivated peanut for high-oleate trait selection. Plant Mol Biol Rep 28:542–548

    Article  CAS  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  Google Scholar 

  • Expósito-Rodríguez M, Borges A, Borges-Pérez A, Pérez J (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:1–131

    Article  Google Scholar 

  • Faccioli P, Ciceri GP, Provero P, Stanca AM, Morcia C, Terzi V (2007) A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies. Plant Mol Biol 63(5):679–688

    Article  PubMed  CAS  Google Scholar 

  • Gachon C, Mingam A, Charrier B (2004) Real-time PCR: what relevance to plant studies? J Exp Bot 55:1445–1454

    Article  PubMed  CAS  Google Scholar 

  • Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30(6):503–512

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008a) 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 6:609–618

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Pelloux J, Bellini C, Van Wuytswinkel O (2008b) Towards a systematic validation of references in real-time RT-PCR. Plant Cell 20(7):1734–1735

    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  Google Scholar 

  • Hu RB, Fan CM, Li HY, Zhang QZ, Fu YF (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10:93

  • 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

  • Iskandar HM, Simpson RS, Casu RE, Bonnett GD, MacLean DJ, Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Rep 22:325–337

    Article  CAS  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of house-keeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Com 345:646–651

    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  Google Scholar 

  • Jiang SJ, Sun YJ, Wang SH (2011) Selection of reference genes in peanut seed by real-time quantitative polymerase chain reaction. Int J Food Sci Technol 46:2191–2196

    Article  CAS  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 

  • Lee JM, Roche JR, Donaghy DJ, Thrush A, Sathish P (2010) Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol 11:8

    Google Scholar 

  • Li QF, Sun SSM, Yuan DY, Yu HX, Gu MH, Liu QQ (2010a) Validation of candidate reference genes for the accurate normalization of real-time quantitative RT-PCR data in rice during seed development. Plant Mol Biol Rep 28:49–57

    Article  Google Scholar 

  • Li MJ, Wang XJ, Su L, Bi YP, Wan SB (2010b) Characterization of five putative acyl carrier protein (ACP) isoforms from developing seeds of Arachis hypogaea L. Plant Mol Biol Rep doi:10.1007/s11105-009-0160-x

  • Libault M, Thibivilliers S, Bilgin D, Radwan O, Benitez M, Clough S, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54

    Article  CAS  Google Scholar 

  • Lin YL, Lai ZX (2010) Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Sci 178:359–365

    Article  CAS  Google Scholar 

  • Luo M, Liang XQ, Dang P, Holbrook CC, Bausher MG, Lee RD, Guo BZ (2005) Microarray-based screening of differentially expressed genes in peanut in response to Aspergillus parasiticus infection and drought stress. Plant Sci 169:695–703

    Article  CAS  Google Scholar 

  • Marino ER, Borges AA, Perez AB, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131

    Article  Google Scholar 

  • Maroufi A, Bockstaele EV, Loose MD (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  PubMed  Google Scholar 

  • Martin RC, Hollenbeck VG, Dombrowski JE (2008) Evaluation of reference genes for quantitative RT-PCR in Lolium perenne. Crop Sci 48:1881–1887

    Article  CAS  Google Scholar 

  • Moore KM, Knauft DA (1989) The inheritance of high-oleic acid in peanut. J Hered 80:252–253

    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 

  • Pan LJ, Yang QL, Chi XY, Chen MN, He YN, Yu SL (2010) AhZFP1, a cDNA encoding C2H2-type zinc finger protein, induced by salt stress in peanut (Arachis hypogaea L.). In: The 4th international conference on bioinformatics and biomedical engineering, pp 978–983

  • 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(1):11

    Article  PubMed  Google Scholar 

  • Perez S, Royo LJ, Astudillo A, Escudero D, Alvarez F, Rodriguez A, Gomez E, Otero J (2007) Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors. BMC Mol Biol 8:114

    Article  PubMed  Google Scholar 

  • Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313(4):856–862

    Article  PubMed  CAS  Google Scholar 

  • Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27

    Article  PubMed  Google Scholar 

  • Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A (2008) Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227(6):1343–1349

    Article  PubMed  CAS  Google Scholar 

  • Ruan W, Lai M (2007) Actin, a reliable marker of internal control? Clin Chim Acta 385:1–5

    Article  PubMed  CAS  Google Scholar 

  • Sakamoto H, Araki T, Meshi T, Iwabuchi M (2000) Expression of a subset of the Arabidopsis Cys2/His2-type zinc-finger protein gene family under water stress. Gene 248:23–32

    Article  PubMed  CAS  Google Scholar 

  • Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136:2734–2746

    Article  PubMed  CAS  Google Scholar 

  • Schmidt GW, Delaney SK (2010) Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283:233–241

    Article  PubMed  CAS  Google Scholar 

  • Suzuki T, Higgins PJ, Crawford DR (2000) Control selection for RNA quantitation. Biotechniques 29:332–333

    PubMed  CAS  Google Scholar 

  • Thorrez L, Van Deun K, Tranchevent LC, Van Lommel L, Engelen K, Marchal K, Moreau Y, Van Mechelen I, Schuit F (2008) Using ribosomal protein genes as reference: a tale of caution. PLoS ONE 3(3):e1854

    Article  PubMed  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

    Google Scholar 

  • Wan HJ, Zhao ZG, Qian CT, Sui YH, Malik AA, Chen JF (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261

    Article  PubMed  CAS  Google Scholar 

  • Yu SL, Pan LJ, Yang QL, Chen MN, Zhang HS (2010) Identification and expression analysis of the phosphoenolpyruvate carboxylase gene family in peanut (Arachis hypogaea L.). Agric Sci China 9(4):477–487

    Article  CAS  Google Scholar 

  • Zhong HY, Chen JW, Li CQ, Chen L, Wu JY, Chen JY, Lu WG, 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 30:641–653

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by grants from China Agriculture Research System (CARS-14), the National Natural Science Foundation of China (31000728; 31100205), the Natural Science Fund of Shandong Province (ZR2009DQ004; ZR2011CQ036), the Promotive Research Fund for Young and Middle-aged Scientists of Shandong Province (BS2010NY023), Qingdao Municipal Science and Technology Plan Project (11-2-4-9-(3)-jch; 11-2-3-26-nsh).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Shandong Peanut Research Institute, Qingdao, 266100, People’s Republic of China

    Xiaoyuan Chi, Qingli Yang, Lijuan Pan, Na Chen, Mingna Chen, Zhen Yang, Tong Wang, Yanan He & Shanlin Yu

  2. Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences (QIBEBT-CAS), Qingdao, 266101, People’s Republic of China

    Ruibo Hu

  3. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People’s Republic of China

    Xiaowen Zhang

Authors
  1. Xiaoyuan Chi
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  2. Ruibo Hu
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  3. Qingli Yang
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  4. Xiaowen Zhang
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  5. Lijuan Pan
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  6. Na Chen
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  7. Mingna Chen
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  8. Zhen Yang
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  9. Tong Wang
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  10. Yanan He
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  11. Shanlin Yu
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Corresponding author

Correspondence to Shanlin Yu.

Additional information

Communicated by S. Hohmann.

X. Chi and R. Hu contributed equally to this work.

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Chi, X., Hu, R., Yang, Q. et al. Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR. Mol Genet Genomics 287, 167–176 (2012). https://doi.org/10.1007/s00438-011-0665-5

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