Scientia Horticulturae 186 (2015) 115–123 Contents lists available at ScienceDirect Scientia Horticulturae journal h om epage: www.elsevier.com/locate/scihorti Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.) Maria Doroteia Campos a , António Miguel Frederico a , Thomas Nothnagel b , Birgit Arnholdt-Schmitt a , Hélia Cardoso a,∗ a EU Marie Curie Chair, ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, IIFA, Universidade de Évora, Ap. 94, 7006-554 Évora, Portugal b Federal Center of Breeding Research on Cultivated Plants, Institute of Horticultural Crops, Neuer Weg 22/23, D-06484 Quedlinburg, Germany a r t i c l e i n f o Article history: Received 7 July 2014 Received in revised form 15 December 2014 Accepted 23 December 2014 Keywords: Reverse transcription quantitative real-time PCR Reference gene Carrot Normalization Gene expression a b s t r a c t Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization through the use of suitable reference genes. This study aimed selecting robust and reliable reference genes which are constitutively and equally expressed for accurate RT- qPCR normalization analysis in two different experimental systems with carrot. A systematic comparison of 12 selected candidate genes for carrot is presented. These included seven genes commonly used as reference gene: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), 18S ribosomal RNA (18S rRNA), ubiquitin (UBQ), alpha-tubulin (TUA), beta-tubulin (ˇ-TUB) and elongation factor-1alpha (EF- 1A). Additionally, other five genes were here presented as potential candidates: other ribosomal RNA (25S and 5.8S rRNA) and ribosomal protein L2 encoding gene (rpL2), the transcriptional initiation factor (TIF1), and the heat shock protein 70 (hsp70). Expression stability was evaluated in: (i) in vivo growth experiment based on carrot tap root secondary growth carried out in growth chambers and (ii) realization phase of somatic embryogenesis. During carrot tap root secondary growth, two reference genes GAPDH and 5.8S rRNA, were stably expressed. In the somatic embryogenesis realization experiment, two ribosomal RNAs were selected as reference genes, the 5.8S and the 25S rRNA. Additionally, the expression profile of the mitochondrial alternative oxidase gene DcAOX1 was conducted in the in vivo growth experiment, to show the impact of reference genes selection. Taken together, our results provide a systematic analysis for the selection of superior reference genes for accurate transcript normalization in carrot, under different experimental conditions. We reinforce the idea that the validation of reference genes for the conditions under study is essential, as emphasized by the expression analysis of DcAOX1 as target gene. These results show that a previous selection of reference genes for each experimental system is crucial to achieve accurate and reliable RT-qPCR gene expression data, avoiding low precision or misleading results. © 2015 Elsevier B.V. All rights reserved. Abbreviations: RT-qPCR, reverse transcription quantitative real time polymerase chain reaction; Ct, cycle threshold; GAPDH, glyceraldehyde 3-phosphate dehydro- genase; TBA, alpha-tubulin; ˇ-TUB, beta-tubulin; ACT, actin; UBQ, ubiquitin; EF-1A, elongation factor 1-alpha; hsp70, heat shock protein 70; rpL2, ribosomal protein L2 encoding gene; TIF1, transcriptional initiation factor; 18S, 25S and 5.8S rRNA, ribo- somal RNAs; M, expression stability; SV, stability value; V, pairwise variation; NF, normalization factor; E, PCR efficiency; AOX, alternative oxidase. ∗ Corresponding author at: EU Marie Curie Chair, ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal. Tel.: +351 2667608000; fax: +351 266760970. E-mail addresses: mdcc@uevora.pt (M.D. Campos), amcmf@uevora.pt (A.M. Frederico), thomas.nothnagel@jki.bund.de (T. Nothnagel), eu chair@uevora.pt (B. Arnholdt-Schmitt), hcardoso@uevora.pt (H. Cardoso). 1. Introduction Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression in various biological systems (Bustin et al., 2005). The accuracy of the results depends strongly on several variables, including the RNA integrity, cDNA synthesis effi- ciency, enzyme and primer performance, reference genes used and method chosen for data analysis (Bustin, 2002; Bustin and Nolan, 2004; Pfaffl, 2001; Skern et al., 2005). All these variables are crucial for relative quantification of gene expression by RT-qPCR. How- ever, because quantification is based on the expression ratio of a target gene versus a reference gene (Pfaffl and Hageleit, 2001), the http://dx.doi.org/10.1016/j.scienta.2014.12.038 0304-4238/© 2015 Elsevier B.V. All rights reserved.