Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Abstract

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…PCR efficiency was determined for each gene by LinRegPCR program (Ramakers et al, 2003;Ruijter et al, 2009). This software was successfully used previously to calculate PCR efficiency (Č ikoš et al, 2007;Feng et al, 2008;Regier & Frey, 2010;Aglawe et al, 2012, Borges et al, 2012.…”

An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR

“…All genes, except for Pv18S, presented SD values lower than one by BestKeeper. For qPCR data normalization, two genes were frequently observed as the optimal number in several experimental plots (Borges et al 2012, Müller et al 2014). …”

“…Establishment of such procedures is not trivial, and efforts to develop seed RNA extraction protocols have been conducted for different species (Christou et al 2014, Ma et al 2015. For the common bean, RNA extraction methods from leaves and roots have been established, which have been based on either commercial kits (Contour-Ansel et al 2010) or not (Borges et al 2012). However, a well-defined RNA extraction protocol for common bean grain/seed tissue is not available and, among legumes, there is just one protocol that was created for soybean seeds (Yin et al 2014).…”

A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development

“…Relative gene expression levels were calculated using the 2 2DCT method, where DCT = CT gene -CT reference gene . Bean Ef1a and IDE were used as internal controls, as described previously (Islas-Flores et al, 2011;Borges et al, 2012). Relative expression values, which were normalized based on two reference genes, were calculated according to the protocol described by Vandesompele et al (2002).…”