title: Detection of alternative splicing during epithelial-mesenchymal transition.
abstract: Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine.
authors.forename: Huilin
authors.initials: H
authors.lastname: Huang
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine; chengc@northwestern.edu.
authors.forename: Chonghui
authors.initials: C
authors.lastname: Cheng
mesh_terms: D017398:Alternative Splicing; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000235:genetics; D042361:Mammary Glands, Human / Q000166:cytology; D062793:Occludin / Q000096:biosynthesis / Q000235:genetics; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014746:Vimentin / Q000096:biosynthesis / Q000235:genetics
chemical_list: C497483:CD44 protein, human; D015820:Cadherins; D005353:Fibronectins; D018960:Hyaluronan Receptors; C568985:OCLN protein, human; D062793:Occludin; D020033:Protein Isoforms; D012333:RNA, Messenger; D014746:Vimentin