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Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects

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Abstract

Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

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References

  • An X-K, Hou Mao-lin, Liu Y-D (2015) Reference gene selection and evaluation for gene expression studies using qRT-PCR in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae). J Econ Entomol 109:879–886

    Article  Google Scholar 

  • Andersen CL, Jensen JL, Ørntoft 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  CAS  PubMed  Google Scholar 

  • Bankowski MJ, Anderson SM (2004) Real-time nucleic acid amplification in clinical microbiology. Clin Microbiol Newsl 26:9–15

    Article  Google Scholar 

  • Brodmann PD, Ilg EC, Berthoud H, Herrmann A (2002) Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food. J AOAC Int 85:646–653

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA et al (2010) MIQE précis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. https://doi.org/10.1186/1471-2199-11-74

    PubMed  PubMed Central  Google Scholar 

  • Cheng D, Zhang Z, He X, Liang G (2013) Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues. PLoS ONE 8:e57718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cusson M (2008) The molecular biology toolbox and its use in basic and applied insect science. Bioscience 58:691–700

    Article  Google Scholar 

  • de Boer ME et al (2009) Reference genes for qRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola). BMC Mol Biol. https://doi.org/10.1186/1471-2199-10-54

    Google Scholar 

  • De Jonge H et al (2007) Evidence based selection of housekeeping genes. PLoS ONE 2:e898

    Article  PubMed  PubMed Central  Google Scholar 

  • de Kok JB et al (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 15:159–185

    Google Scholar 

  • Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechnology 37:112–119

    CAS  Google Scholar 

  • Feuer R, Vlaic S, Arlt J, Sawodny O, Dahmen U, Zanger UM, Thomas M (2015) LEMming: A linear error model to normalize parallel quantitative real-time PCR (qPCR) data as an alternative to reference gene based methods. PLoS ONE 10:e0135852

    Article  PubMed  PubMed Central  Google Scholar 

  • Fu W et al (2013) Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae). Int J Biol Sci 9:792–802

    Article  PubMed  PubMed Central  Google Scholar 

  • Galiveti CR, Rozhdestvensky TS, Brosius J, Lehrach H, Konthur Z (2010) Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR. RNA 16:450–461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guénin 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. Exp Bot 60:487–493

    Article  Google Scholar 

  • Hellemans J, Mortier G, DePaepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genom Biol. https://doi.org/10.1186/gb-2007-8-2-r19

    Google Scholar 

  • Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal Bioanal Chem 375:985–993

    Article  CAS  PubMed  Google Scholar 

  • Hsiao L-L et al (2001) A compendium of gene expression in normal human tissues. Physiol Genom 7:97–104

    Article  CAS  Google Scholar 

  • Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284

    Article  CAS  PubMed  Google Scholar 

  • Jung M, Ramankulov A, Roigas J, Johannsen M, Ringsdorf M, Kristiansen G, Jung K (2007) In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR. BMC Mol Biol. https://doi.org/10.1186/1471-2199-8-47

    PubMed  PubMed Central  Google Scholar 

  • Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl Genet 54:391–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kubista M et al (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125

    Article  CAS  PubMed  Google Scholar 

  • Li R et al (2013) Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS ONE 8:e53006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Losey JE, Vaughan M (2006) The economic value of ecological services provided by insects. Bioscience 56:311–323

    Article  Google Scholar 

  • Lu Y, Yuan M, Gao X, Kang T, Zhan S, Wan H, Li J (2013) Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae). PLoS ONE 8:e68059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lu Y et al (2015) Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens (Lepidoptera: Noctuidae). J Asia-Pac. Entomol 18:669–675

    Article  CAS  Google Scholar 

  • Mamidala P, Rajarapu SP, Jones SC, Mittapalli O (2011) Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius. J Med Entomol 48:947–951

    Article  CAS  PubMed  Google Scholar 

  • Niu J, Cappelle K, de Miranda JR, Smagghe G, Meeus I (2014) Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris. J Invert Pathol 115:76–79

    Article  CAS  Google Scholar 

  • Nonis A, De Nardi B, Nonis A (2014) Choosing between RT-qPCR and RNA-seq: a back-of-the-envelope estimate towards the definition of the break-even-point. Anal Bioanal Chem 406:3533–3536

    CAS  PubMed  Google Scholar 

  • Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A (2014) A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Det Quant 1:23–33

    Google Scholar 

  • Park Y, Kim J, Choi JR, Song J, Chung JS, Lee K-A (2008) Evaluation of multiplex PCR assay using dual priming oligonucleotide system for detection mutation in the Duchenne muscular dystrophy gene. Korean J Lab Med 28:386–391

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) AZ of quantitative PCR. International University Line (IUL), La Jolla, pp 87–212

    Google Scholar 

  • Ponton F, Chapuis M-P, Pernice M, Sword GA, Simpson SJ (2011) Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Ins Physiol 57:840–850

    Article  CAS  Google Scholar 

  • Popovici V, Goldstein DR, Antonov J, Jaggi R, Delorenzi M, Wirapati P (2009) Selecting control genes for RT-QPCR using public microarray data. BMC Bioinform. https://doi.org/10.1186/1471-2105-10-42

    Google Scholar 

  • Postollec F, Falentin H, Pavan S, Combrisson J, Sohier D (2011) Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiol 28:848–861

    Article  CAS  PubMed  Google Scholar 

  • Radonić 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 313:856–862

    Article  PubMed  Google Scholar 

  • Rhinn H, Marchand-Leroux C, Croci N, Plotkine M, Scherman D, Escriou V (2008) Housekeeping while brain’s storming validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury. BMC Mol Biol. https://doi.org/10.1186/1471-2199-9-62

    PubMed  PubMed Central  Google Scholar 

  • Rodrigues TB et al (2014) Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera). PLoS ONE 9:e109825

    Article  PubMed  Google Scholar 

  • Rodríguez A, Rodríguez M, Luque MI, Justesen AF, Córdoba JJ (2011) Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods. Int J Food Microbiol 149:226–235

    Article  PubMed  Google Scholar 

  • Rodríguez A, Rodríguez M, Córdoba JJ, Andrade MJ (2015) Design of primers and probes for quantitative real-time PCR methods. PCR Primer Des 1275:31–56

    Article  Google Scholar 

  • Rodríguez-Lázaro D, Hernández M (2013) Real-time PCR in food science: introduction. Curr Issues Mol Biol 15:25–38

    PubMed  Google Scholar 

  • Scharlaken B, de Graaf DC, Goossens K, Brunain M, Peelman LJ, Jacobs FJ (2008) Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. J Ins Sci. https://doi.org/10.1673/031.008.3301

    Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108

    Article  CAS  PubMed  Google Scholar 

  • Shakeel M, Zhu X, Kang T, Wan H, Li J (2015) Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J Asia-Pac Entomol 18:123–130

    Article  CAS  Google Scholar 

  • Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. https://doi.org/10.1186/1471-2199-7-33

    PubMed  PubMed Central  Google Scholar 

  • Sobhy H, Colson P (2012) Gemi: PCR primers prediction from multiple alignments. Comp Funct Genomics. https://doi.org/10.1155/2012/783138

    PubMed  PubMed Central  Google Scholar 

  • Spinsanti G, Panti C, Lazzeri E, Marsili L, Casini S, Frati F, Fossi CM (2006) Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Mol Biol. https://doi.org/10.1186/1471-2199-7-32

    PubMed  PubMed Central  Google Scholar 

  • Suda T, Katoh M, Hiratsuka M, Fujiwara M, Irizawa Y, Oshimura M (2003) Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene. Int J Mol Med 12:243–246

    CAS  PubMed  Google Scholar 

  • Sun M, Lu M-X, Tang X-T, Du Y-Z (2015) Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae). PLoS ONE 10:e0115979

    Article  PubMed  PubMed Central  Google Scholar 

  • Suzuki T, Higgins P, Crawford D (2000) Control selection for RNA quantitation. Biotechnology 29:332–337

    CAS  Google Scholar 

  • Teng X, Zhang Z, He G, Yang L, Li F (2012) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. J Insect Sci. https://doi.org/10.1673/031.012.6001

    PubMed  PubMed Central  Google Scholar 

  • Thorrez L et al (2008) Using ribosomal protein genes as reference: a tale of caution. PLoS ONE 3:e1854

    Article  PubMed  PubMed Central  Google Scholar 

  • Valasek MA, Repa JJ (2005) The power of real-time PCR. Adv Physiol Educ 29:151–159

    Article  PubMed  Google Scholar 

  • van Doorn R, Szemes M, Bonants P, Kowalchuk GA, Salles JF, Ortenberg E, Schoen CD (2007) Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™. BMC Genom. https://doi.org/10.1186/1471-2164-8-276

    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. Genom Biol 3:0034

    Article  Google Scholar 

  • Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N (eds) Real-time PCR: current technology and applications. Caister Academic Press, London, pp 47–64

    Google Scholar 

  • Wang X, Seed B (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res 31:e154–e154

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang GH, Xia QY, Cheng DJ, Duan J, Zhao P, Chen J, Zhu L (2008) Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR. Ins Sci 15:405–413

    Article  Google Scholar 

  • Waxman S, Wurmbach E (2007) De-regulation of common housekeeping genes in hepatocellular carcinoma. BMC Genom. https://doi.org/10.1186/1471-2164-8-243

    Google Scholar 

  • Yang Q et al (2014) Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria (Orthoptera: Acrididae). PLoS ONE 9:e98164

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang C, Pan H, Noland JE, Zhang D, Zhang Z, Liu Y, Zhou X (2015) Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific Rep. https://doi.org/10.1038/srep18201

    Google Scholar 

  • Yuan M et al (2014) Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR. PLoS ONE 9:e86503

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang S et al (2015) Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae). Gene 555:393–402

    Article  CAS  PubMed  Google Scholar 

  • Zheng Y-T, Li H-B, Lu M-X, Du Y-Z (2014) Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae). PLoS ONE 9:e111369

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhong M, Wang X, Wen J, Cai J, Wu C, Aly SM (2013) Selection of reference genes for quantitative gene expression studies in the house fly (Musca domestica L.) using reverse transcription quantitative real-time PCR. Acta Biochim Biophys Sin 45:1067–1073

    Article  Google Scholar 

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Acknowledgements

This work is supported by the National Natural Science Foundation of China (31572069, 31371989). We thank the reviewers for their constructive comments to improve our manuscript. We apologize to those scientists whose work was not cited in this manuscript owing to space limitations.

Supporting Information

Supplementary Table 1—List of reference genes used in insect studies

Supplementary Table 2—Traditional reference genes showing low stability under different biotic and abiotic conditions, specifically in insects, published in important scientific journals.

Supplementary Table 3—Reference genes (traditional and novel) showing high stability under different biotic conditions, specifically in insects, published in important scientific journals.

Supplementary Table 4—Reference genes (traditional and novel) showing high stability under different abiotic conditions, specifically in insects, published in important scientific journals.

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Authors and Affiliations

  1. Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of Agriculture, South China Agricultural University, Guangzhou, China

    Muhammad Shakeel & Fengliang Jin

  2. Faculty of Veterinary Science, Food Hygiene and Safety, Meat and Meat Products Research Institute, University of Extremadura, Avda. de la Universidad, s/n, 10003, Cáceres, Spain

    Alicia Rodriguez

  3. Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, China

    Urfa Bin Tahir

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  1. Muhammad Shakeel
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  2. Alicia Rodriguez
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  3. Urfa Bin Tahir
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  4. Fengliang Jin
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Correspondence to Fengliang Jin.

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Shakeel, M., Rodriguez, A., Tahir, U.B. et al. Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects. Biotechnol Lett 40, 227–236 (2018). https://doi.org/10.1007/s10529-017-2465-4

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