Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR.
OBJECTIVE
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.
Wang
W H
WH
Alcon Research, Ltd., Fort Worth, TX 76134, USA.
McNatt
L G
LG
Shepard
A R
AR
Jacobson
N
N
Nishimura
D Y
DY
Stone
E M
EM
Sheffield
V C
VC
Clark
A F
AF
D000368:Aged; D000369:Aged, 80 and over; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D004268:DNA-Binding Proteins; D005123:Eye / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D051858:Forkhead Transcription Factors; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D005901:Glaucoma / Q000151:congenital* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014019:Tissue Donors; D014021:Tissue Preservation; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; D014157:Transcription Factors; D012313:RNA