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

Wan-Heng Wang; L G McNatt; N Jacobson; D Y Nishimura; Allan R Shepard; E M Stone; V C Sheffield; A F Clark

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

Journal article

Open access

Peer reviewed

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

Wan-Heng Wang, L G McNatt, N Jacobson, D Y Nishimura, Allan R Shepard, E M Stone, V C Sheffield and A F Clark

Molecular vision, Vol.7(13), pp.89-94

04/17/2001

PMID: 11320352

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Abstract

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. 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. 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. 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.

Gene Expression

Humans

Middle Aged

Male

DNA Primers - chemistry

Tissue Distribution

RNA - isolation & purification

Aged, 80 and over

Female

Glaucoma - metabolism

DNA-Binding Proteins

RNA - metabolism

DNA, Complementary - analysis

Eye - metabolism

Tissue Preservation

Eye - chemistry

Gene Expression Profiling - methods

Transcription Factors - genetics

Reverse Transcriptase Polymerase Chain Reaction

Glaucoma - congenital

Transcription Factors - metabolism

Aged

Tissue Donors

Glaucoma - genetics

Forkhead Transcription Factors

Details

Title: Subtitle: Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR
Creators: Wan-Heng Wang
L G McNatt
N Jacobson
D Y Nishimura - University of Iowa, Ophthalmology and Visual Sciences
Allan R Shepard
E M Stone - University of Iowa, Theatre Arts
V C Sheffield - University of Iowa, Stead Family Department of Pediatrics
A F Clark
Resource Type: Journal article
Publication Details: Molecular vision, Vol.7(13), pp.89-94
PMID: 11320352
NLM abbreviation: Mol Vis
ISSN: 1090-0535
eISSN: 1090-0535
Publisher: United States
Grant note: R01-EY-10564 / NEI NIH HHS
Language: English
Date published: 04/17/2001
Academic Unit: Stead Family Department of Pediatrics; Iowa Neuroscience Institute; Medical Genetics and Genomics; Ophthalmology and Visual Sciences
Record Identifier: 9983979952302771

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