Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in SD-2a.

The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. (), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (, and ) were analyzed to determine the most stable reference genes for RT-qPCR in SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v) ethanol). The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that and were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, and would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of , and would be suitable for 12% ethanol shock treatment. and revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in SD-2a under ethanol stress conditions.