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Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp.

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 experimental 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. Normalization 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.

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Acknowledgments

Financial support was provided by “Fundaçãopara a Ciência e a Tecnologia” (FCT) (Project PTDC/AGR-AAM/64078/2006) and by the European Fund FEDER. A.S. Fortunato acknowledges a postdoctoral grant from FCT (SFRH/BPD/47563/2008). The authors acknowledge Isabel Palos and Sandra Anjos for excellent technical support.

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Correspondence to Luis F. Goulao.

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Luis F. Goulao and Ana S. Fortunato contributed equally to this work.

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Online Resource Fig. S1

Confirmation of primer specificity of studied genes in qRT-PCR amplifications. Melting curves generated for all genes, showing the presence of a single peak and lack of non-specific fragments or primer dimers. Identical traces were obtained for the other two genotypes (not shown) (PPT 166 kb)

Online Resource Fig. S2

Gene expression stability and ranking of the ten reference genes as predicted, automatically, by geNorm. Plots are based on mean expression stability (M values) calculated in (a) the total assay, (b) the genotype series, (c) in the drought stress assay, (d) in the cold stress, and (e) in the multiple stress assay. V values were obtained using geNorm as the pairwise variation (V n / n + 1) between NF n and NF n + 1, based on the ranked genes, according to the stepwise exclusion of the least stable genes and M value recalculation, as implemented in the algorithm (PPT 392 kb)

Online Resource Table S1

BestKeeper software statistics for the ten reference genes based on Ct values in the experimental series studied (XLS 53 kb)

Online Resource Table S2

Stability values for the best combination of two reference genes, based on the NormFinder output (XLS 34 kb)

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Goulao, L.F., Fortunato, A.S. & C. Ramalho, J. Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp.. Plant Mol Biol Rep 30, 741–759 (2012). https://doi.org/10.1007/s11105-011-0382-6

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Keywords

  • Abiotic stress
  • Coffea
  • Gene expression normalization
  • Quantitative real-time PCR
  • Reference genes