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Background  Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained.  Toona ciliata  Roem ( T. ciliata ). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.    Results  The experimental results showed that  TUB-α  was the most stably expressed reference gene across all samples and  UBC17  was the most stable in leaves and young stems under  Hypsipyla robusta  ( H. robusta ) and methyl jasmonate (MeJA) treatments. In addition,  PP2C59  and  UBC5B  were the best-performing genes in leaves under  H. robusta  treatment, while  HIS1  and  ACT7  were the best reference genes in young stems. The two best reference genes were  60S-18  and  TUB-α  after treatment at 4 °C. The expression of  HIS6  and  MUB1  was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 ( TcMYB3)  gene.    Conclusions  This is the first report to verify the best reference genes for normalizing gene expression in  T. ciliata  under different conditions, which will facilitate future elucidation of gene regulations in this species.

bmc plant biology

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis