Jump to Main Content
Evaluation of reference genes for normalizing RT-qPCR in leaves and suspension cells of Cephalotaxus hainanensis under various stimuli
- Sun, Huapeng, Jiang, Xuefei, Sun, Mengli, Cong, Hanqing, Qiao, Fei
- Plant methods 2019 v.15 no.1 pp. 31
- Cephalotaxus, algorithms, gene expression, genes, leaves, medicinal plants, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, reverse transcription, transcriptome
- BACKGROUND: Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used approach for investigating gene expression levels in plants because of its high reproducibility, sensitivity, accuracy and rapidness. Evaluation of reference genes for normalizing RT-qPCR data is a necessary step, especially in new plant varieties. Cephalotaxus hainanensis is a precious medicinal plant belonging to the family of Cephalotaxaceae and no RT-qPCR studies have been reported on it. RESULTS: In this study, 9 candidate reference genes were selected from the transcriptome data of C. hainanensis; 3 statistical algorithms (geNorm, NormFinder, BestKeeper) were applied to evaluate their expression stabilities through 180 samples under 6 stimuli treatments in leaves and leaf-derived suspension cultured cells; a comprehensive stabilities ranking was also performed by RefFinder. The results showed that suitable reference genes in C. hainanensis should be selected for normalization relative to different experimental sets. 18S showed a higher stability than other candidate reference genes which ranked at the top two suitable genes under all experimental setups in this study. CONCLUSION: This study is the first to evaluate the stability of reference genes in C. hainanensis and supply an important foundation to use the RT-qPCR for an accurate and far-reaching gene expression analysis in C. hainanensis.