Abstract
The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data.
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Acknowledgements
The authors thank the members of the Laboratory of Plant Molecular Physiology at the Universidade Federal de Lavras for their collaboration to this work and the Fundação Procafé for providing plant material to this study.
Funding
We also thank the National Institute of Science and Technology of Coffee (INCT-Café) for providing funding and the National Council for Scientific and Technological Development (CNPq), the Minas Gerais Research Foundation (FAPEMIG), and the Coordination of Improvement of Higher Education (CAPES) for grants.
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Online Resource 1
The RNA Integrity Number (RIN) Electrophoresis File Run Summary (Chip Summary) of the biological triplicates of RNA samples from WD Roots, WD Stems, WD Leaves and WW Roots of Coffea arabica. (PDF 256 kb)
Online Resource 2
The RNA Integrity Number (RIN) Electrophoresis File Run Summary (Chip Summary) of the biological triplicates of RNA samples from WW Stems, WW Leaves, WW Flowers and WW Fruits of Coffea arabica. (PDF 255 kb)
Online Resource 3
The RNA Integrity Number (RIN) Electrophoresis File Run Summary (Chip Summary) of the biological triplicates of RNA samples from WW Roots, WW Stems, WW Leaves and WD Flowers of Coffea canephora. (PDF 257 kb)
Online Resource 4
The RNA Integrity Number (RIN) Electrophoresis File Run Summary (Chip Summary) of the biological triplicates of RNA samples from WW Fruits of Coffea canephora. (PDF 254 kb)
Online Resource 5
Panel of the dissociation curves (Melt curves) of the primers of the candidate genes tested in Coffea arabica samples. The panels are named after the respective gene. The unique peaks show specificity of the primers. (GIF 3804 kb)
Online Resource 6
Panel of the dissociation curves (Melt curves) of the primers of the candidate genes tested in Coffea canephora samples. The panels are named after the respective gene. The unique peaks show specificity of the primers. (GIF 2213 kb)
Online Resource 7
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for all tissues grouped of C. arabica (GIF 30 kb)
Online Resource 8
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for root of C. arabica under WW and WD conditions (GIF 30 kb)
Online Resource 9
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for stem of C. arabica under WW and WD conditions (GIF 27 kb)
Online Resource 10
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for leaf of C. arabica under WW and WD conditions (GIF 28 kb)
Online Resource 11
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for leaf and root of C. arabica under WW and WD conditions (GIF 30 kb)
Online Resource 12
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for all tissues grouped of C. arabica under WD conditions (GIF 28 kb)
Online Resource 13
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for root of C. arabica under WD conditions (GIF 27 kb)
Online Resource 14
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for stem of C. arabica under WD conditions (GIF 25 kb)
Online Resource 15
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for leaf of C. arabica under WD conditions (GIF 24 kb)
Online Resource 16
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for all tissues grouped of C. arabica under WW conditions (GIF 28 kb)
Online Resource 17
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for root of C. arabica under WW conditions (GIF 25 kb)
Online Resource 18
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for stem of C. arabica under WW conditions (GIF 26 kb)
Online Resource 19
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for leaf of C. arabica under WW conditions (GIF 25 kb)
Online Resource 20
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for flower of C. arabica under WW conditions (GIF 23 kb)
Online Resource 21
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for fruit of C. arabica under WW conditions (GIF 25 kb)
Online Resource 22
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for all tissues grouped of C. canephora under WW conditions (GIF 27 kb)
Online Resource 23
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for root of C. canephora under WW conditions (GIF 24 kb)
Online Resource 24
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for stem of C. canephora under WW conditions (GIF 23 kb)
Online Resource 25
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for leaf of C. canephora under WW conditions. (GIF 21 kb)
Online Resource 26
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for flower of C. canephora under WW conditions (GIF 21 kb)
Online Resource 27
Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn + 1 values were used for decision of the optimal number of reference genes for fruit of C. canephora under WW conditions (GIF 21 kb)
Online Resource 28
RefFinder ranking for all conditions and combinations tested in C. arabica tissues. (XLSX 1211 kb)
Online Resource 29
RefFinder ranking for all conditions and combinations tested in C. canephora tissues. (XLSX 734 kb)
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Fernandes-Brum, C.N., Garcia, B.d.O., Moreira, R.O. et al. A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions. Tree Genetics & Genomes 13, 131 (2017). https://doi.org/10.1007/s11295-017-1213-1
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DOI: https://doi.org/10.1007/s11295-017-1213-1