Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR.

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  • Additional Information
    • Source:
      Publisher: Molecular Vision Country of Publication: United States NLM ID: 9605351 Publication Model: Electronic Cited Medium: Internet ISSN: 1090-0535 (Electronic) Linking ISSN: 10900535 NLM ISO Abbreviation: Mol Vis Subsets: MEDLINE
    • Publication Information:
      Original Publication: Atlanta Ga : Molecular Vision, 1995-
    • Subject Terms:
      DNA-Binding Proteins*; Eye/*chemistry ; Glaucoma/*congenital ; Glaucoma/*genetics ; RNA/*isolation & purification ; Transcription Factors/*genetics; Aged ; Aged, 80 and over ; DNA Primers/chemistry ; DNA, Complementary/analysis ; Eye/metabolism ; Female ; Forkhead Transcription Factors ; Gene Expression ; Gene Expression Profiling/methods ; Glaucoma/metabolism ; Humans ; Male ; Middle Aged ; RNA/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Distribution ; Tissue Donors ; Tissue Preservation ; Transcription Factors/metabolism
    • Abstract:
      Purpose: To develop methods for obtaining high quality RNA from human donor eyes and to determine the expression profile of the congenital glaucoma gene FOXC1 in human ocular tissues.
      Methods: To obtain high quality RNA from donor eyes, several different preservation methods were tested including storing eyes on ice, freezing eyes, and placing eyes in the commercial fixative RNAlaterTM prior to dissection and RNA extraction. Nine different ocular tissues from human donors were dissected and examined. Pigment-free total RNA was isolated and used for quantitative real-time RT-PCR using FOXC1 and GAPDH (internal standard) primers to assess the quality and expression of FOXC1.
      Results: An expression profile of FOXC1 in human ocular tissues was determined using quantitative PCR of RNA isolated using a simple and effective procedure for ocular tissue preservation and pigment-free RNA isolation. Higher quality RNA was obtained from human donor eyes preserved in RNAlaterTM compared to RNA extracted from eyes stored on ice or frozen at -80 degrees C. RNA extraction techniques that removed interfering pigment from ocular tissues produced RNA that could be easily amplified by PCR. In the adult human eye, expression of FOXC1 was greatest in the trabecular meshwork (TM) followed by the optic nerve head, choroid/RPE, ciliary body, cornea, and iris. FOXC1 expression levels were much lower in other non-ocular human tissues, such as liver, muscle, lung, heart, and kidney.
      Conclusions: Using an optimized donor eye preservation method and tissue RNA isolation procedure, we show that the FOXC1 transcription factor gene, which is known to be associated with developmental glaucoma, also may have an important role in the adult eye.
    • Grant Information:
      R01-EY-10564 United States EY NEI NIH HHS
    • Accession Number:
      0 (DNA Primers)
      0 (DNA, Complementary)
      0 (DNA-Binding Proteins)
      0 (FOXC1 protein, human)
      0 (Forkhead Transcription Factors)
      0 (Transcription Factors)
      63231-63-0 (RNA)
    • Publication Date:
      Date Created: 20010426 Date Completed: 20010823 Latest Revision: 20071114
    • Publication Date:
      20221213
    • Accession Number:
      11320352