Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

Abstract

Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.

Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

“…The coefficient of variation (CV) for the overall assay was 5.35% (total assay variability), which is within the range (from 3.4% to 11.6%) of values reported in previously for qPCR (Pfaffl et al, 2004). Overall, the X-fold variance values in this study are somewhat higher than the values that have been observed in other plant systems (e.g., Mukesh et al, 2006;Remans et al, 2008, Barsalobres-Cavallari et al, 2009. Besides laboratory working errors these higher values may indicate high levels of variability across peanut organs and tissues, and this variability may be partially explained by the fact that many housekeeping genes, like gapdh, are not only important for basal cell metabolism, but also participate in other cellular functions (Singh and Green, 1993;Ishitani et al, 1996).…”

“…In Arachis, for example, the ubiquitin gene is sometimes used in qPCR studies as a stable housekeeping gene (e.g., Luo et al, 2005;Nobile et al, 2007). However, several reports have shown that the expression levels of these so-called housekeeping genes differ among different plant tissues and organs (Jain et al, 2006;Remans et al, 2008;Barsalobres-Cavallari et al, 2009). Furthermore, normalization to a single housekeeping gene can lead to invalid normalization, because many housekeeping genes, are not only important for basal cell metabolism, but also participate in other cellular functions that may vary under changing conditions (Singh and Green, 1993).…”

“…In plants, a growing number of studies have evaluated reference genes in detail, including studies in rice (Jain et al, 2006;Mukesh et al, 2006), poplar (Brunner et al, 2004), potato (Nicot et al, 2005), soybean (Jian et al, 2008), Arabidopsis (Remans et al, 2008), grapevine (Reid et al, 2006), and Coffea arabica (Barsalobres-Cavallari et al, 2009). The search for the most stable reference gene usually involves scoring the stability of a series of 8-15 candidate genes for a set of different tissues and/or conditions.…”

Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought

“…Various studies of different plant species have defined reference genes for different organs (Nicot et al, 2005;Jain et al, 2006;Gutierrez et al, 2008;Hu et al, 2009;Qi et al, 2010;Manoli et al, 2012) or different environmental conditions (Nicot et al, 2005;Reid et al, 2006;Barsalobres-Cavallari et al, 2009;Hu et al, 2009). However, few studies regarding the validation of reference genes in rice have been reported (Kim et al, 2003;Jain et al, 2006;Narsai et al, 2010;Wang et al, 2010).…”

Real Time PCR: the Use of Reference Genes and Essential Rules Required to Obtain Normalisation Data Reliable to Quantitative Gene Expression

“…Several reference genes, including those coding for biological products such as tubulins, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphatases, albumin, cyclophilin, micro-globulin, ribosomal units (18S rRNA) or ubiquitin (UBQ) have been described in the literature (Foss et al, 2003;Rocha et al, 2013). The correct choice of reference genes is crucial to properly analyze the results of qPCR (Suzuki et al, 2000) and to measure and reduce the errors from variations among the samples (Barsalobres-Cavallari et al, 2009). …”