Reliable Reference Genes for Accurate Gene Expression Profiling across Different Tissues and Genotypes of Rice Seedlings ( Oryza sativa L.) under Salt Stress

Gene expression regulation is one of the most effective adaptation responses to abiotic stressors. Quantitative real-time polymerase chain reaction (RT-qPCR) is the method of choice for quantifying gene expression levels. Reference genes are important factors that are required for accurate and reliable normalization of RT-qPCR-derived data, and a minimum of two stably expressed reference genes must be employed according to the Minimum Information for publication of Quantitative Real-Time PCR Experiment guidelines. To date, most gene expression studies reported in rice under salt stress have utilized a single reference gene. In addition, there has been little research into a set of reference genes that are stably expressed across tissues of different rice genotypes. In this study, twelve potential reference gene candidates were selected, including ACT11 , TIP41 , BTUB , EF1A , EIF4A , FBOX , GAPDH , PP2A , SPX , UBCE2 , UBQ10 , and CCZ1 . Their expression stability was evaluated in internode, leaf, and root tissues of six rice genotypes with different salt stress tolerances. The geNorm, NormFinder, and RefFinder statistical algorithms identified EIF4A and TIP41 as the most suitable set of reference genes for the accurate and reliable normalization of RT-qPCR data generated from eighteen tissue samples. The performance of the identified reference genes was validated for their accuracy and reliability through RT-qPCR analysis of the gene encoding the HKT1 potassium transporter. Our results highlighted the importance of identifying a suitable set of reference genes for gene expression studies in rice under salt stress in order to obtain accurate and reliable data.