Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp. Luis F. Goulao & Ana S. Fortunato & José C. Ramalho Published online: 23 December 2011 # Springer-Verlag 2011 Abstract Selection and validation of appropriate reference genes should be the first step to consider in experiments based on quantitative real-time polymerase chain reaction (qRT-PCR). In this study, ten candidate genes were investigated for their stability as suitable reference genes in qRT-PCR data normalization using a diverse set of 12 Coffea cDNAs from plants from three different species/ genotypes exposed to single or multiple abiotic stresses (drought and chilling, alone or in combination). Primer amplification efficiencies were calculated for all of the selected genes and varied according to each individual genotype. The expression of each gene was measured by qRT-PCR to evaluate its stability. A multiple analytical approach was followed, based on consensus merged data from four different complementary statistics, namely geNorm, BestKeeper, NormFinder, and coefficient of variation, which produced comparable but not identical results. According to this approach, the most suitable sets of reference genes for data normalization in the five experi- mental datasets are (1) total assay: GAPHD, Cycl, and UBQ10; (2) genotype: GAPDH, UBQ10, Ap47, and EF-1A; (3) cold stress: UBQ10, GAPDH, ACT, and EF-1A; (4) drought stress: GAPDH, ACT, EF1A, and Apt; and (5) multiple stress: UBQ10, GAPDH, ACT, and elf-4A. Nor- malization of gene expression using these selected genes was validated by examination of the expression of the photosynthetic-related ApoA2 gene in samples from non- stressed and stressed plants. Our results are useful to assist studies on Coffea physiology with the aim of breeding for increased tolerance to abiotic stress conditions. Keywords Abiotic stress . Coffea . Gene expression normalization . Quantitative real-time PCR . Reference genes Introduction Coffee is a major crop of the world’s agricultural sector, supporting the economy of many countries from tropical regions. To assure its viability and sustainability, the use of genotypes with the ability to cope with environmental constraints is mandatory. Previous reports have highlighted that, within the Coffea genus, stress responses to energy overpressure related to cold, drought, and high irradiance conditions implicate acclimation changes that promote the tolerance of the photosynthetic apparatus (Ramalho et al. 1999, 2000, 2003; Batista-Santos et al. 2011) due to improved membrane stability and to the reinforcementof the anti-oxidative system (Fortunato et al. 2010; Lima et al. 2002; Partelli et al. 2009; Pinheiro et al. 2004; Ramalho et al. 1998). These differences to survive in environmental limiting areas make breeding for inter-species hybrids a promising strategy to explore in order to obtain plants adapted to a variety of climates and ecological units with regular productions and high yields. To accomplish such goal, it is fundamental to acquire knowledge on the physiological basis of the distinct adaptation mechanisms and to identify molecular cues associated with abiotic stress tolerance. Luis F. Goulao and Ana S. Fortunato contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11105-011-0382-6) contains supplementary material, which is available to authorized users. L. F. Goulao (*) : A. S. Fortunato : J. C. Ramalho Centro de Ecofisiologia, Bioquímica e Biotecnologia Vegetal, Instituto de Investigação Científica Tropical I.P. (IICT, IP), Av. da República, Quinta do Marquês, 2784-505 Oeiras, Portugal e-mail: goulao@iict.pt Plant Mol Biol Rep (2012) 30:741–759 DOI 10.1007/s11105-011-0382-6