Biologia plantarum 62:89-96, 2018 | DOI: 10.1007/s10535-017-0754-3

Assessment of reference genes for real-time quantitative PCR normalization in Ilex paraguariensis leaves during drought

R. M. Acevedo1, E. H. Avico1, O. A. Ruiz2, P. A. Sansberro1,*
1 Laboratorio de Biotecnología Aplicada y Genómica Funcional, Instituto de Botánica del Nordeste, Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Corrientes, Argentina
2 Unidad de Biotecnología 1, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Chascomús, Buenos Aires, Argentina

Reverse transcription of RNA followed by real-time quantitative PCR (qPCR) is to date the most reliable method for gene expression studies. However, to control the errors introduced along the numerous experimental procedures, it requires a normalization using internal reference genes with stable expression. To address this issue, nine candidate reference genes were investigated in Ilex paraguariensis leaves subjected to water stress. To facilitate the selection, we analysed the real-time qPCR data with three different software programs. The obtained results support the conclusion that RNA polymerase associated protein rtf1 homolog (RTF) combined with any of the following pairs is the most suitable triad of genes to compute a normalization factor: elongation factor 1-alpha + tubulin alpha chain (EF1a + α-Tub), actin + cyclophilin 38 (ACT + CYP38), or cyclophilin 38 + vacuolar protein sorting-associated protein 18 homologs (CYP38 + VPS). Our analysis constitutes the first in-depth study to identify the appropriate reference genes for the quantification of transcription in Ilex paraguariensis leaves during drought and provides essential information for further gene expression studies in this tree species.

Keywords: abiotic stresses; gene expression; house-keeping genes
Subjects: reference genes; PCR normalization; expression stability; drought stress; waterlogging

Received: February 8, 2017; Revised: April 26, 2017; Accepted: May 11, 2017; Published: January 1, 2018Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Acevedo, R.M., Avico, E.H., Ruiz, O.A., & Sansberro, P.A. (2018). Assessment of reference genes for real-time quantitative PCR normalization in Ilex paraguariensis leaves during drought. Biologia plantarum, 62(1), 89-96. doi: 10.1007/s10535-017-0754-3.
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    Supplementary files

    Download filebpl-201801-0009_S1.pdf

    File size: 975.65 kB

    References

    1. Acevedo, R.M., Maiale, S.J., Pessino, S.C., Bottini, R., Ruiz, O.A., Sansberro, P.A.: A succinate dehydrogenase flavoprotein subunit-like transcript is upregulated in Ilex paraguariensis leaves in response to water deficit and abscisic acid. - Plant Physiol. Biochem. 65: 48-54, 2013. Go to original source...
    2. Acevedo, R.M., Ruiz, O.A., Sansberro, P.A.: Gene expression changes in response to drought stress in Ilex paraguariensis leaves. - Plant Omics 9: 334-343, 2016. Go to original source...
    3. Andersen, C.L., Jensen, J.L., Ørntoft, T.F.: 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, 2004. Go to original source...
    4. Brunner, A.M., Yakovlev, I.A., Strauss, S.H.: Validating internal controls for quantitative plant gene expression studies. - BMC Plant Biol. 4: 14, 2004. Go to original source...
    5. Czechowski, T., Bari, R.P., Stitt, M., Scheible, W.R., Udvardi, M.K.: Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root-and shoot-specific genes. - Plant J. 38: 366-379, 2004. Go to original source...
    6. Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., Scheible, W.R.: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. - Plant Physiol. 139: 5-17, 2005. Go to original source...
    7. Derveaux, S., Vandesompele, J., Hellemans, J.: How to do successful gene expression analysis using real-time PCR. - Methods 50: 227-230, 2010. Go to original source...
    8. Die, J.V., Román, B.: RNA quality assessment: a view from plant qPCR studies. - J. exp. Bot. 63: 6069-6077, 2012. Go to original source...
    9. Faccioli, P., Ciceri, G.P., Provero, P., Stanca, A.M., Morcia, C., Terzi, V.: 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: 679-688, 2007. Go to original source...
    10. Gantasala, N.P., Papolu, P.K., Thakur, P.K., Kamaraju, D., Sreevathsa, R., Rao, U.: Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L). - BMC Res. Notes 6: 312, 2013. Go to original source...
    11. Gottlieb, A., Giberti, G., Poggio, L.: Molecular analyses of the genus Ilex (Aquifoliaceae) in southern South America, evidence from AFLP and its sequence data. - Amer. J. Bot. 92: 352-369, 2005. Go to original source...
    12. Gutierrez, L., Mauriat, M., Guénin, S., Pelloux, J., Lefebvre, J.F., Louvet, R., Rusterucci, C., Moritz, T., Guerineau, F., Bellini, C., Van Wuytswinkel, O.: 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, 2008. Go to original source...
    13. Heck, C.I., De Mejia, E.G.: Yerba mate tea (Ilex paraguariensis): a comprehensive review on chemistry, health implications, and technological considerations. - J. Food Sci. 72: R138-R151, 2007. Go to original source...
    14. Huggett, J., Dheda, K., Bustin, S., Zumla, A.: Real-time RT-PCR normalisation; strategies and considerations. - Genes Immun. 6: 279-284, 2005. Go to original source...
    15. Kakar, K., Wandrey, M., Czechowski, T., Gaertner, T., Scheible, W.R., Stitt, M., Torres-Jerez, I., Xiao, Y., Redman, J.C., Wu, H.C., Cheung, F., Town, C.D., Udvardi, M.K.: A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula. - Plant Methods 4: 18, 2008. Go to original source...
    16. Kim, B.R., Nam, H.Y., Kim, S.U., Kim, S.I., Chang, Y.J.: Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. - Biotechnol. Lett. 25: 1869-1872, 2003.
    17. Kim, S.G., Lee, J.S., Kim, J.T., Kwon, Y.S., Bae, D.W., Bae, H.H., Son, B.Y., Baek, S.B., Kwon, Y.U., Woo, M.O., Shin, S.: Physiological and proteomic analysis of the response to drought stress in an inbred Korean maize line. - Plant Omics 8: 159-168, 2015.
    18. Kozera, B., Rapacz, M.: Reference genes in real-time PCR. - J. appl. Genet. 54: 391-406, 2013. Go to original source...
    19. Kumar, V., Sharma, R., Trivedi, P.C., Vyas, G.K., Khandelwal, V.: Traditional and novel references towards systematic normalization of qRT-PCR data in plants. - Aust. J. Crop Sci. 5: 1455-1468, 2011.
    20. Lee, J.M., Roche, J.R., Donaghy, D.J., Thrush, A., Sathish, P.: Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). - BMC mol. Biol. 11: 8, 2010. Go to original source...
    21. Lefever, S., Hellemans, J., Pattyn, F., Przybylski, D.R., Taylor, C., Geurts, R., Untergasser, A., Vandesompele, J.: RDML: structured language and reporting guidelines for real-time quantitative PCR data. - Nucl. Acids Res. 37: 2065-2069, 2009. Go to original source...
    22. Liang, P., Pardee, A.B.: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. - Science 257: 967-971, 1992. Go to original source...
    23. Lima, M.E., Colpo, A.C., Salgueiro, W.G., Sardinha, G.E., Ávila, D.S., Folmer, V.: Ilex paraguariensis extract increases lifespan and protects against the toxic effects caused by paraquat in Caenorhabditis elegans. - Int. J. environ. Res. Public Health 11: 10091-10104, 2014. Go to original source...
    24. Maroufi, A.: Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress. - Biol. Plant. 60: 645-654, 2016. Go to original source...
    25. Nicot, N., Hausman, J.F., Hoffmann, L., Evers, D.: Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. - J. exp. Bot. 56: 2907-2914, 2005. Go to original source...
    26. Paolacci, A.R., Tanzarella, O.A., Porceddu, E., Ciaffi, M.: Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. - BMC mol. Biol. 10: 11, 2009. Go to original source...
    27. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP.: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. - Biotechnol. Lett. 26: 509-515, 2004. Go to original source...
    28. Rasmussen, R.: Quantification on the LightCycler. - In Meuer, S., Wittwer, C., Nakagawara, K.I. (ed.): Rapid Cycle Realtime PCR, Methods and Applications. Pp 21-34. Springer-Verlag, Berlin - Heidelberg 2001.
    29. Reid, K.E., Olsson, N., Schlosser, J., Peng, F., Lund, S.T.: 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, 2006. Go to original source...
    30. Sansberro, P.A., Mroginski, L.A., Bottini, R.: Abscisic acid promotes Ilex paraguariensis growth by alleviating diurnal water stress. - Plant Growth Regul. 42: 105-111, 2004. Go to original source...
    31. Shinozaki, K., Yamaguchi-Shinozaki, K.: Gene networks involved in drought stress response and tolerance. - J. exp. Bot. 58: 221-227, 2007.
    32. Singh, D., Laxmi, A.: Transcriptional regulation of drought response: a tortuous network of transcriptional factors. - Front. Plant Sci. 6: 895, 2015. Go to original source...
    33. Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A., Heinen, E.: Housekeeping genes as internal standards: Use and limits. - J. Biotechnol. 75: 291-295, 1999. Go to original source...
    34. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F.: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. - Genome Biol. 3: Research0034, 2002.
    35. Wan, H., Zhao, Z., Qian, C., Sui, Y., Malik, A.A., Chen, J.: Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. - Anal. Biochem. 399: 257-261, 2010. Go to original source...
    36. Zhu, J., Zhang, L., Li, W., Han, S., Yang, W., Qi, L.: Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. - PLoS ONE 8: e53196, 2013. Go to original source...

    Return to the content