Abstract
Expression analyses such as quantitative and/or real-time PCR require the use of reference genes for normalization in order to obtain reliable assessments. The expression levels of these reference genes must remain constant in all different experimental conditions and/or tissues under study. Traditionally, housekeeping genes have been used for this purpose, but most of them have been reported to vary their expression levels under some experimental conditions. Consequently, the election of the best reference genes should be tested and validated in every experimental scenario. Microarray data are not always available for the search of appropriate reference genes, but NGS experiments are increasingly common. For this reason, an automatic workflow based on mapped NGS reads is presented with the aim of obtaining putative reference genes for a giving species in the experimental conditions of interest. The calculation of the coefficient of variation (CV) and a simple, normalized expression value such as RPKM per transcript allows for filtering and selecting those transcripts expressed homogeneously and consistently in all analyzed conditions. This workflow has been tested with Roche/454 reads obtained from olive (Olea europaea L.) pollen and pistil at different developmental stages, as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana. Some of the putative candidate reference genes have been experimentally validated.
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References
Freeman, W.M., Walker, S.J., Vrana, K.E.: Quantitative RT-PCR: pitfalls and potential. Biotechniques 26, 112–125 (1999)
Lee, P.D., Sladek, R., Greenwood, C.M.T., Hudson, T.J.: Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res. 12, 292–297 (2002)
Suzuki, T., Higgins, P.J., Crawford, D.R.: Control selection for RNA quantitation. Biotechniques 29, 332–337 (2000)
Brunner, A.M., Yakovlev, I.A., Strauss, S.H.: Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol. 4, 14 (2004)
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K.: Genome-wide identification and testing of superior reference genes for transcript normalization. Society 139, 5–17 (2005)
Nonis, A., Vezzaro, A., Ruperti, B.: Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (olea europaea L.) fruits. J. Agric. Food Chem. 60, 6855–6865 (2012)
Resetic, T., Stajner, N., Bandelj, D., Javornik, B., Jakse, J.: Validation of candidate reference genes in RT-qPCR studies of developing olive fruit and expression analysis of four genes involved in fatty acids metabolism. Mol. Breed. 32, 211–222 (2013)
Ray, D.L., Johnson, J.C.: Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR. BMC Res. Notes. 7, 304 (2014)
Ma, J., Skibbe, D.S., Fernandes, J., Walbot, V.: Male reproductive development: gene expression profiling of maize anther and pollen ontogeny. Genome Biol. 9, R181 (2008)
Seoane, P., Carmona, R., Bautista, R., Guerrero-Fernández, D., Claros, G.: AutoFlow: an easy way to build workflows. In: Proceedings IWBBIO, pp. 342–349 (2014)
Lutz, U., Posé, D., Pfeifer, M., Gundlach, H., Hagmann, J., Wang, C., Weigel, D., Mayer, K.F.X., Schmid, M., Schwechheimer, C.: Modulation of Ambient Temperature-Dependent Flowering in Arabidopsis thaliana by Natural Variation of FLOWERING LOCUS M. PLoS Genet. 11, e1005588 (2015)
Carmona, R., Zafra, A., Seoane, P., Castro, A.J., Guerrero-Fernández, D., Castillo-Castillo, T., Medina-García, A., Cánovas, F.M., Aldana-Montes, J.F., Navas-Delgado, I., de Alché, J.D., Claros, M.G.: ReprOlive: a database with linked data for the olive tree (Olea europaea L.) reproductive transcriptome. Front. Plant Sci. 6, 625 (2015)
Lamesch, P., Berardini, T.Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., Muller, R., Dreher, K., Alexander, D.L., Garcia-Hernandez, M., Karthikeyan, A.S., Lee, C.H., Nelson, W.D., Ploetz, L., Singh, S., Wensel, A., Huala, E.: The Arabidopsis Information Resource (TAIR): Improved gene annotation and new tools. Nucleic Acids Res. 40, 1202–1210 (2012)
Falgueras, J., Lara, A.J., Fernández-Pozo, N., Cantón, F.R., Pérez-Trabado, G., Claros, M.G.: SeqTrim: a high-throughput pipeline for pre-processing any type of sequence read. BMC Bioinformatics 11, 1–12 (2010)
Langmead, B., Salzberg, S.L.: Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012)
Goto, N., Prins, P., Nakao, M., Bonnal, R., Aerts, J., Katayama, T.: BioRuby: Bioinformatics software for the Ruby programming language. Bioinformatics 26, 2617–2619 (2010)
de Dios, A.J., Castro, A.J., Olmedilla, A., Fernández, M.C., Rodríguez, R., Villalba, M., Rodríguez-García, M.I.: The major olive pollen allergen (Ole e I) shows both gametophytic and sporophytic expression during anther development, and its synthesis and storage takes place in the RER. J. Cell Sci. 112, 2501–2509 (1999)
Coker, J.S., Davies, E.: Selection of candidate housekeeping controls in tomato plants using EST data. Biotechniques 35, 740–748 (2003)
Vezzaro, A., Krause, S.T., Nonis, A., Ramina, A., Degenhardt, J., Ruperti, B.: Isolation and characterization of terpene synthases potentially involved in flavor development of ripening olive (Olea europaea) fruits. J. Plant Physiol. 169, 908–914 (2012)
Yanik, H., Turktas, M., Dundar, E., Hernandez, P., Dorado, G., Unver, T.: Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.). BMC Plant Biol. 13, 10 (2013)
Nicot, N.: Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J. Exp. Bot. 56, 2907–2914 (2005)
Maroufi, A., Van Bockstaele, E., De Loose, M.: Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol. Biol. 11, 15 (2010)
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)
Die, J.V., Román, B., Nadal, S., González-Verdejo, C.I.: Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232, 145–153 (2010)
Ronning, C.M., Stegalkina, S.S., Ascenzi, R.A., Bougri, O., Hart, A.L., Utterbach, T.R., Vanaken, S.E., Riedmuller, S.B., White, J.A., Cho, J., Pertea, G.M., Lee, Y., Karamycheva, S., Sultana, R., Tsai, J., Quackenbush, J., Griffiths, H.M., Restrepo, S., Smart, C.D., Fry, W.E., Van Der Hoeven, R., Tanksley, S., Zhang, P., Jin, H., Yamamoto, M.L., Baker, B.J., Buell, C.R.: Comparative analyses of potato expressed sequence tag libraries. Plant Physiol. 131, 419–429 (2003)
Gohain, B.: Rubisco-bis-phosphate oxygenase (RuBP)- A potential housekeeping gene forqPCR assays in tea. African J. Biotechnol. 11, 11193–11199 (2012)
Hoedemaekers, K., Derksen, J., Hoogstrate, S.W., Wolters-Arts, M., Oh, S.-A., Twell, D., Mariani, C., Rieu, I.: BURSTING POLLEN is required to organize the pollen germination plaque and pollen tube tip in Arabidopsis thaliana. New Phytol. 206, 255–267 (2015)
Acknowledgments
This work has been supported by co-funding from the ERDF (European Regional Development Fund) and (i) MINECO (grants BFU2011-22779 and RECUPERA2020-3.1.4.), (ii) INIA (grant RTA2013-00068-C03-02), and (iii) PAI (grants P10-CVI-6075, P10-AGR-6274 and P11-CVI-7487). The authors also thankfully acknowledge the computer resources and the technical support provided by the Plataforma Andaluza de Bioinformática of the University of Málaga.
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Carmona, R., Seoane, P., Zafra, A., Jiménez-Quesada, M.J., Alché, J.d.D., Claros, M.G. (2016). Automatic Workflow for the Identification of Constitutively-Expressed Genes Based on Mapped NGS Reads. In: Ortuño, F., Rojas, I. (eds) Bioinformatics and Biomedical Engineering. IWBBIO 2016. Lecture Notes in Computer Science(), vol 9656. Springer, Cham. https://doi.org/10.1007/978-3-319-31744-1_36
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