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

Casuarina glauca is a model actinorhizal plant species that establishes N₂-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.