Detection of alternative splicing during epithelial-mesenchymal transition

J Vis Exp. 2014 Oct 9;(92):e51845. doi: 10.3791/51845.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Alternative Splicing
  • Cadherins / biosynthesis
  • Cadherins / genetics
  • Epithelial Cells / cytology
  • Epithelial Cells / physiology*
  • Epithelial-Mesenchymal Transition / genetics*
  • Fibronectins / biosynthesis
  • Fibronectins / genetics
  • Humans
  • Hyaluronan Receptors / biosynthesis
  • Hyaluronan Receptors / genetics
  • Mammary Glands, Human / cytology
  • Occludin / biosynthesis
  • Occludin / genetics
  • Protein Isoforms
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Vimentin / biosynthesis
  • Vimentin / genetics


  • CD44 protein, human
  • Cadherins
  • Fibronectins
  • Hyaluronan Receptors
  • OCLN protein, human
  • Occludin
  • Protein Isoforms
  • RNA, Messenger
  • Vimentin