Optimal procedure for extracting RNA from human ocular tissuesand expression profiling of the congenital glaucoma gene FOXC1 usingquantitative RT-PCR

[摘要] Purpose: To develop methods for obtaining high quality RNA fromhuman donor eyes and to determine the expression profile of thecongenital glaucoma gene FOXC1 in human ocular tissues.Methods: To obtain high quality RNA from donor eyes, severaldifferent preservation methods were tested including storing eyes onice, freezing eyes, and placing eyes in the commercial fixativeRNAlaterTM prior to dissection and RNA extraction. Nine differentocular tissues from human donors were dissected and examined.Pigment-free total RNA was isolated and used for quantitative real-timeRT-PCR using FOXC1 and GAPDH (internal standard) primers to assessthe quality and expression of FOXC1.Results: An expression profile of FOXC1 in human ocular tissueswas determined using quantitative PCR of RNA isolated using a simple andeffective procedure for ocular tissue preservation and pigment-free RNAisolation. Higher quality RNA was obtained from human donor eyespreserved in RNAlaterTM compared to RNA extracted from eyes storedon ice or frozen at -80 °C. RNA extraction techniques that removedinterfering pigment from ocular tissues produced RNA that could beeasily amplified by PCR. In the adult human eye, expression of FOXC1was greatest in the trabecular meshwork (TM) followed by the optic nervehead, choroid/RPE, ciliary body, cornea, and iris. FOXC1 expressionlevels 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 andtissue RNA isolation procedure, we show that the FOXC1 transcriptionfactor gene, which is known to be associated with developmentalglaucoma, also may have an important role in the adult eye.

[学科分类] 生物化学/生物物理