Skip to main content
SpringerLink
Log in

References genes for qRT-PCR in guaraná (Paullina cupana var. sorbilis)

  • Published:
Brazilian Journal of Botany Aims and scope Submit manuscript

Abstract

Gene expression has been extensively studied in plant science research, mainly for the assessment of plant stress responses. Real-time-quantitative polymerase chain reaction (RT-qPCR) is an important tool for obtaining this information because it is a quick and easy technique to acquire a large amount of molecular data for both model and non-model plants. For a successful RT-qPCR analysis, gene expression should be carefully normalised. Genes involved in essential biological processes that exhibit constitutive expression are commonly selected as internal standards to normalise RT-qPCR experiments. In this study, the transcription profiles of 13 candidate reference genes for RT-qPCR were evaluated in three guarana cultivars (BRS-Amazonas, BRS-Maués and BRS-Luzéia) using different tissues (vegetative and fruit) in varying developmental stages. Two different algorithms, NormFinder and GeNorm, were utilised to assess gene stability. In general, the two algorithms did not select the same pairs of genes for all analysed conditions. For the largest group (the fruits of all cultivars), NormFinder selected the pair EF1A/UBQ, whereas GeNorm chose ACT/GAPDH as the best normalising genes. Thus, we recommend the use of at least four reference genes for the normalisation of gene expression in guarana plant studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Aerts JL, Gonzales MI, Topalian SL (2004) Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR. Biotechniques 36:84–97

    CAS  PubMed  Google Scholar 

  • Albertino SMF, Nascimento Filho FJ, da Silva JF, Atroch AL, Galvão AKDL (2012) Rooting of guarana cultivar cuttings with fertilization of matrix plants. Pesqui Agropecu Bras 47:1449–1454

    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 

  • Ângelo PCS et al (2008) Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome. Plant Cell Rep 27:117–124

    Article  PubMed  Google Scholar 

  • Atroch AL (2009) Avaliação e seleção de progênies de meios irmãos de guaranazeiro (Paullinia cupana var. sorbilis (Mart.) Ducke) utilizando caracteres morfo-agronômicos. Thesis, Instituto Nacional de Pesquisas da Amazônia -INPA

  • Barsalobres-Cavallari CF, Severino FE, Maluf MP, Maia IG (2009) Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol 10:1

    Article  PubMed Central  PubMed  Google Scholar 

  • Bentes JLS, Costa Neto PQ (2011) Variability of Colletotrichum guaranicola using AFLP markers. Acta Amazon 41:251–256

    Article  Google Scholar 

  • Borges A, Tsai S, Caldas DG (2012) Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses. Plant Cell Rep 31:827–838

    Article  CAS  PubMed  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 Central  PubMed  Google Scholar 

  • Chandna R, Augustine R, Bisht N (2012) Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One. doi:10.1371/journal.pone.0036918

    PubMed Central  PubMed  Google Scholar 

  • Conrath U (2011) Molecular aspects of defence priming. Trends Plant Sci 16:524–531

    Article  CAS  PubMed  Google Scholar 

  • Cruz F et al (2009) Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breed 23:607–616

    Article  CAS  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi M, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17. doi:10.1104/pp.105.063743

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dheda K, Huggett J, Bustin S, Johnson M, Rook G, Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37:112

    CAS  PubMed  Google Scholar 

  • Ding J, Jia J, Yang L, Wen H, Zhang C, Liu W, Zhang D (2004) Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. J Agric Food Chem 52:3372–3377

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  PubMed  Google Scholar 

  • Fernández-Aparicio M et al (2013) Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development. Mol Biol Rep 40:3395–3407

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  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. J Exp Bot 60:487–493

    Article  PubMed  Google Scholar 

  • Han X, Lu M, Chen Y, Zhan Z, Cui Q, Wang Y (2012) Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. PLoS One. doi:10.1371/journal.pone.0043084

    Google Scholar 

  • Hao DC, Chen SL, Xiao PG, Liu M (2012) Application of high-throughput sequencing in medicinal plant transcriptome studies. Drug Dev Res 73:487–498

    Article  CAS  Google Scholar 

  • Heard CM, Johnson S, Moss G, Thomas CP (2006) In vitro transdermal delivery of caffeine, theobromine, theophylline and catechin from extract of guarana, Paullinia cupana. Int J Pharm 317:26–31

    Article  CAS  PubMed  Google Scholar 

  • 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 housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651

    Article  CAS  PubMed  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 Central  PubMed  Google Scholar 

  • Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10:4

    Article  PubMed Central  PubMed  Google Scholar 

  • Marta F, Carlos V, Cristián B, Sofia V (2010) Validation of reference genes for real-time qRT-PCR normalization during cold acclimation in Eucalyptus globulus. Trees 24:1109–1116

    Article  Google Scholar 

  • Moura J, Araújo P, Brito M, Souza U, Viana J, Mazzafera P (2012) Validation of reference genes from Eucalyptus spp. under different stress conditions. BMC Res Notes. doi:10.1186/1756-0500-5-634

    Google Scholar 

  • Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (2014) The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold and heat. Front Plant Sci. doi:10.3389/fpls.2014.00170

    PubMed Central  PubMed  Google Scholar 

  • Nascimento Filho FJ, Atroch AL, Cruz CD, Carneiro PCS (2009) Adaptabilidade e estabilidade de clones de guaraná. Pesq Agropec Bras 44:1138–1144

    Article  Google Scholar 

  • Oliveira L, Breton M, Bastolla F, Camargo S, Margis R, Frazzon J, Pasquali G (2012) Reference genes for the normalization of gene expression in Eucalyptus species. Plant Cell Physiol 53:405–422

    Article  PubMed  Google Scholar 

  • Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: best Keeper–excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515

    Article  CAS  PubMed  Google Scholar 

  • Podevin N, Krauss A, Henry I, Swennen R, Remy S (2012) Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa. Mol Breed 30:1237–1252

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rajeevan MS, Vernon SD, Taysavang N, Unger ER (2001) Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J Mol Diagn 3:26–31

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rasmussen R (2001) Quantification on the lightcycler. In: Wittwer C, Hahn M, Kaul K (eds) Rapid cycle real-time PCR. Springer, New York, pp 21–34

    Chapter  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 227:1343–1349

    Article  CAS  PubMed  Google Scholar 

  • Rezaian M, Krake L (1987) Nucleic acid extraction and virus detection in grapevine. J Virol Methods 17:277–285

    Article  CAS  PubMed  Google Scholar 

  • Schimpl FC, da Silva JF, Gonçalves JFC, Mazzafera P (2013) Guarana: revisiting a highly caffeinated plant from the Amazon. J Ethnopharmacol 150:14–31

    Article  CAS  PubMed  Google Scholar 

  • Schimpl FC, Kiyota E, Mayer JLS, Gonçalves JFC, da Silva JF, Mazzafera P (2014) Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit. Phytochemistry 105:25–36

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Thellin O et al (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291–295

    Article  CAS  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. doi:10.1186/gb-2002-3-7-research0034

    PubMed Central  PubMed  Google Scholar 

  • Wei L, Miao H, Zhao R, Han X, Zhang T, Zhang H (2013) Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR. Planta 237:873–889

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yan J, Yuan F, Long G, Qin L, Deng Z (2012) Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Mol Biol Rep 39:1831–1838

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FCS - 2011/03266-6 and APDJ - Fapesp) and Conselho Nacional Científico e Tecnológico (PM) for the doctoral and research fellowships, respectively.

Author information

Authors and Affiliations

  1. Departamento de Biologia Vegetal-IB, CP 6109, Universidade Estadual de Campinas, Campinas, São Paulo, 13083-970, Brazil

    Flávia Camila Schimpl, Adilson Pereira Domingues Júnior & Paulo Mazzafera

  2. Instituto Nacional de Pesquisas da Amazônia, Manaus, Amazonas, Brazil

    José Francisco de Carvalho Gonçalves

  3. Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas, Brazil

    José Ferreira da Silva

Authors
  1. Flávia Camila Schimpl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adilson Pereira Domingues Júnior
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José Francisco de Carvalho Gonçalves
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. José Ferreira da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paulo Mazzafera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paulo Mazzafera.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schimpl, F.C., Domingues Júnior, A.P., de Carvalho Gonçalves, J.F. et al. References genes for qRT-PCR in guaraná (Paullina cupana var. sorbilis). Braz. J. Bot 38, 281–288 (2015). https://doi.org/10.1007/s40415-015-0147-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40415-015-0147-9

Keywords

Search

Navigation