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

Abstract

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 droughtstressed 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.

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

“…However, in most plant species reference genes have only been validated for a single cultivar. Cruz et al (2009) [3] examined using potential reference genes with leaves of three different coffee (Coffea arabica) cultivars. The stability ranking of candidate reference genes based on results obtained using multiple cultivars was very different than rankings observed based on an examination of different organs of a single cultivar or stressed leaves or roots of a single cultivar.…”

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

“…A reference gene should have stable expression in different organs, developmental stages, and environments. According to previous works on the best reference genes for transcription normalization in plants, the most reliable are those constitutively expressed and involved in basic cellular processes, such as protein and sugar metabolism and maintenance of cell structure (Cruz et al, 2009).…”

Expression of endogenous proteins in maize hybrids in a multi-location field trial in India

“…Understanding the factors that contribute to the variability in concentration of these housekeeping proteins may provide insight to explain the variability that is expected for transgenic proteins in GM crops. Actin, EF1-alpha and GAPDH were selected as example proteins for assessment because they are well studied (Brunner Cruz et al 2009;Gimeno et al 2014;Gutha et al 2010;Li et al 2011;Paolacci et al 2009), and indirect quantitative ELISA methods could be developed with commercially available antibodies. In this study, the concentrations of all three endogenous proteins were influenced by growth stage, hybrid background, and location, to some extent.…”