Selection of Reference Genes for RT-qPCR Studies in Different Organs of Rice Cultivar BRS AG Submitted to Recurrent Saline Stress

Quantitative real-time polymerase chain reactions (RT-qPCR) have become one of the most widely used methods for analyzing gene expression, provided suitable reference genes are available to normalize the data. RNA was isolated from leaves, grain, rachises and sheaths of rice ( Oryza sativa L. cv. BRS AG) submitted to different saline stress events for seven days, and expression analysis was carried out by RT-qPCR. Expression levels of ten candidate reference genes were assessed, actin11 ( ACT11 ), ubiquitin conjugating enzyme E2 ( UBC-E2 ), eukaryotic elongation factor1- α ( Eef-1 α), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), β -tubulin (β -Tub ) , eukaryotic initiation factor 4a ( Eif-4- α), ubiquitin10 ( UBQ10 ), ubiquitin5 ( UBQ5 ), aquaporin TIP41 ( TIP41-like ). Gene expression stability was calculated using the common statistical algorithms geNorm, BestKeeper and ΔCt method, NormFinder and RefFinder. The most stably expressed genes were UBC2E and GAPDH for leaves, UBQ5 and UBQ10 for sheaths, TIP41 and UBQ10 for rachises, and TIP41 and cyclophilin for grain. Gene expression of triose phosphate translocator ( TPT1) , ADP-glucose transporter ( BT1-1 ), choline monooxygenase (CMO) was used to validate the selected reference genes. The results highlighted the importance of using suitable reference gene to normalize gene expression data in rice plants.