title: Validation and application of reference genes for quantitative gene expression analyses in various tissues of Bupleurum chinense.
abstract: It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.
authors.affiliation: Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Hai Dian District, Beijing 100193, China.
authors.forename: Lemeng
authors.initials: L
authors.lastname: Dong
authors.forename: Chun
authors.initials: C
authors.lastname: Sui
authors.forename: Yujun
authors.initials: Y
authors.lastname: Liu
authors.forename: Yun
authors.initials: Y
authors.lastname: Yang
authors.forename: Jianhe
authors.initials: J
authors.lastname: Wei
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Yang
mesh_terms: D029972:Bupleurum / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D009928:Organ Specificity / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D012984:Software; D014158:Transcription, Genetic
chemical_list: D010940:Plant Proteins; D012333:RNA, Messenger