Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

Abstract

Casuarina glauca is a model actinorhizal plant species that establishes N 2 -fixing symbiosis with Frankia bacteria. This plant is highly resilient to extreme environments, being commonly found in saline zones. Gene expression studies by quantitative real-time polymerase chain reaction (qRT-PCR) constitute a powerful tool to analyze the mechanisms underlying plant stress-tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to produce accurate data. In this work we report on the evaluation of a set of ten reference genes to be used in qRT-PCR studies in C. glauca grown under high salt concentrations, following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Five independent methods (geNorm, NormFinder, BestKeeper, Coefficient of Variance, and ReFinder) were used to evaluate gene stability. According to the results, the calibration of qRT-PCR reactions with the most versus the least stable reference genes produced different expression patterns of C. glauca stress responsive genes (CgCS and CgAPX). The same was observed when data was normalized with one, two or three stable reference genes. These study constitutes a baseline for accurate qRT-PCR analysis in C. glauca exposed to high salt concentrations which should include the use of at least two stable reference genes.

Disentangling the role of extracellular polysaccharides in desiccation tolerance in lichen‐forming microalgae. First evidence of sulfated polysaccharides and ancient sulfotransferase genes

“…The amplification efficiency of each set of primers was confirmed as being higher than 95% according to the standard curve method described by da Costa et al . (2015). Transcript quantification was normalized to Ap47 (Clathrin adaptor complexes subunit) and Act (Actin) as reference genes following the strategy proposed by Vandesompele et al .…”

Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

“…Therefore, it is essential to screen suitable reference genes in different tissues of H. perforatum under different experimental conditions. Ribosomal RNA and some HKGs are usually used as reference genes, such as actin ( ACT ), tubulin ( TUB) , glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), and polyubiquitin ( UBQ ) (Costa et al, 2015; Goulao, Fortunato & Ramalho, 2012; Llanos, François & Parrou, 2015; Willems, Leyns & Vandesompele, 2008), whereas many studies have revealed that the most commonly used HKGs are not always reliable among different experimental samples. Thus, an evaluation to screen the optimal HKGs in different species is urgently needed (Ohl et al, 2005; Selvey et al, 2001; Thellin et al, 1999).…”

Selection and Validation of Reference Genes for Accurate RT-qPCR Data Normalization in Coffea spp. under a Climate Changes Context of Interacting Elevated [CO2] and Temperature

“…To avoid severe pitfalls in data analysis and interpretation, this implies the selection and systematic validation of suitable reference genes to be used as internal controls. In fact, expression stability should be validated for each particular plant tissue/organ, cell, and experimental conditions, particularly when involving environmental stressful conditions (Die et al, 2010 ; El Kelish et al, 2014 ; Imai et al, 2014 ; da Costa et al, 2015 ). In addition, the stability of reference gene transcripts is also species-dependent (Andersen et al, 2004 ; Gutierrez et al, 2008a , b ), as in the case of coffee (Cruz et al, 2009 ; Goulao et al, 2012 ).…”

“…In addition, the stability of reference gene transcripts is also species-dependent (Andersen et al, 2004 ; Gutierrez et al, 2008a , b ), as in the case of coffee (Cruz et al, 2009 ; Goulao et al, 2012 ). A number of housekeeping genes (e.g., b-actin, elongation factor1a, 18S ribosomal RNA, and polyubiquitin) involved in general cell metabolism pathways are widely used to calibrate RT-qPCR studies in biological systems (Willems et al, 2008 ; Goulao et al, 2012 ; Imai et al, 2014 ; da Costa et al, 2015 ; Llanos et al, 2015 ). However, their expression levels may vary with the experimental conditions (Petriccione et al, 2015 ), and their use as internal reference genes should be taken with caution (Gutierrez et al, 2008a , b ).…”