title: Technical brief: Optimized pipeline for isolation of high-quality RNA from corneal cell subpopulations.
abstract: OBJECTIVE
  Attempts to determine the transcriptional profile of discrete subsets of limbal epithelial cells in situ using laser capture microdissection (LCM) face two major challenges. First, the transcriptional profile of cells within a tissue may rapidly change as the tissue is excised and exposed to cold ischemia. Second, there is a risk of degradation of the RNA as the cellular compartment is separated from the remaining tissue. An optimized protocol for LCM of corneal epithelium is presented to address these issues.
  
  
  METHODS
  Experiments using porcine eye globes were carried out to determine both optimal procedures and settings for tissue harvest, transport, storage, histology, LCM, and RNA isolation. The optimized protocol was validated using human corneal epithelium.
  
  
  RESULTS
  To facilitate preservation of the gene expression profile, we have developed a mechanical tool for dissection of cornea that, in combination with flash freezing, enables tissue to be stored within 5 min of enucleation of the eye. Furthermore, we describe how RNA from limbal crypt cells may be obtained using a procedure involving cryosectioning, histological staining, and LCM.
  
  
  CONCLUSIONS
  In this paper, we describe an optimized method for isolating high-quality RNA from cellular subpopulations confined to the limbal crypts of the cornea. The procedure yields RNA in amounts and quality suitable for downstream gene expression analyses, such as microarrays or next generation sequencing.
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark ; Department of Ophthalmology, Aalborg University Hospital, Denmark.
authors.forename: Chris
authors.initials: C
authors.lastname: Bath
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark.
authors.forename: Trine
authors.initials: T
authors.lastname: Fink
authors.affiliation: Department of Ophthalmology, Aalborg University Hospital, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Vorum
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Hjortdal
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Zachar
mesh_terms: D000818:Animals; D003315:Cornea / Q000166:cytology* / Q000378:metabolism*; D004867:Equipment Design; D005615:Freezing; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods* / Q000592:standards*; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification* / Q000592:standards*; D034421:Sus scrofa
chemical_list: D012313:RNA