Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

• Published in: Molecular breeding Vol. 23; no. 4; pp. 607 - 616
• Main Authors: Cruz, Fernanda, Kalaoun, Samara, Nobile, Paula, Colombo, Carlos, Almeida, Juliana, Barros, Leila M. G, Romano, Eduardo, Grossi-de-Sá, Maria Fátima, Vaslin, Maité, Alves-Ferreira, Marcio
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.05.2009; Springer Netherlands; Springer Nature B.V
• Subjects: Agricultural commodities; Agronomy; Algorithms; Biomedical and Life Sciences; Biotechnology; Coffea; Coffea arabica; Coffee; Cultivars; Drought; Gene expression; Genes; Genetics; Glyceraldehyde-3-phosphate dehydrogenase; guidelines; Leaves; Life Sciences; Molecular biology; Northern blotting; Organs; Plant biology; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Plant species; Polymerase chain reaction; quantitative polymerase chain reaction; Real time; roots; water stress; Real-time PCR; Reference genes; Drought stress; Development; qPCR; Coffee; Gene expression
• ISSN: 1380-3743; 1572-9788
• DOI: 10.1007/s11032-009-9259-x

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here.