Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress

Suitable reference gene (RGs) is the prerequisite for accurate normalization of real-time quantitative PCR (RT-qPCR) data. However, previous results are diverse in various researches that focused on selecting stable RGs. This study aims at systematically assessing various RGs in plants under salt stress or drought stress by collection of geNorm rankings of genes, data transformation and statistic analysis. Although none of the analyzed genes can guarantee universally stable expressions in plant species under salt stress or drought stress, we found that 18S (18S ribosomal RNA) was generally the least stable gene under salt and drought stress. This gene should not be used as the RG in RT-qPCR. On the contrary, it is least risk to use EF1 for salt stress and TIP41 for drought treatment experiments. We compared the effects of salt and drought stresses on 7 frequently used RGs through paired-samples T test. The expression of Ubiquitin gene under drought stress is much more unstable than that under salt stress. The tested genes belonging to multi-gene family and having different stability could be one reason of variations in the published studies, which was supported by the analysis of expression profile of Salicornia europaea transcriptome. This is the first systematic assessment quantifying global stability of RGs across plant species under salt stress and drought stress, which will improve our understanding of RGs and facilitate the future work on RGs selection.