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C. Chen, K. Malik, T.W. Olsen; Stability of RNA From the Retina and Retinal Pigment Epithelium in a Porcine Model Simulating Human Eye-Bank Conditions . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2289.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To assess RNA stability post-mortem using a porcine model to simulate current human eye bank techniques. Methods: Eye bank time interval data was collected from 191 donor specimens: death to refrigeration, enucleation, and tissue processing. A control porcine eye was enucleated, retina and RPE isolated, and specimens frozen. Fourteen porcine eyes remained at room temperature for 2 hours, then cooled on ice. Retina and RPE were isolated and frozen at 5, 12, 24, 29, 36, 48, and 72 hours. Four globes remained in moist chamber, 5 whole and 5 sectioned globes were immersed in RNAlater at either 5, 12, 24, or 48 hours. RNA was isolated. The 28S and 18S rRNA peaks were analyzed by electrophoresis. RT-PCR was performed on each sample. Messenger RNA for GAPDH, Actin, mRHO (from retina), and RPE-65 (from RPE) were analyzed with gel electrophoresis. Results: The average time (hours) from death to refrigeration is 4.2, to enucleation 6.4, and tissue processing 10.7. RT-PCR gel electrophoresis patterns from retinal tissue had isointense bands at each interval from Actin, GAPDH, and mRHO. Band patterns from RPE demonstrated decay of the RT-PCR gene products after 5 hours. This decay was delayed by at least 24 hours with the use of RNAlater. The 28S rRNA decay is similar for retina and RPE. Conclusions: Retinal tissue RNA can be analyzed within the time constraints of current eye bank tissue processing, whereas analysis of RPE necessitates either rapid processing or use of RNAlater. These results should aid in future studies that utilize eye bank tissue for RNA analysis.
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