Stability of RNA from the retina and retinal pigment epithelium in a porcine model simulating human eye bank conditions.
OBJECTIVE
To assess RNA stability after death in a porcine model to simulate current human eye bank techniques.


METHODS
Eye bank time interval data were 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 (-80 degrees C). Fourteen porcine eyes remained at room temperature for 2 hours and then cooled to 4 degrees C. Retina and RPE were isolated and frozen (-80 degrees C) at 5, 12, 24, 29, 36, 48, and 72 hours. Four globes remained in a moist chamber, five whole and five sectioned globes were immersed in RNAlater (Ambion, Austin, TX) at 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, beta-actin, mouse rhodopsin from retina (mRHO), and RPE-65 (from RPE) were analyzed with gel electrophoresis.


RESULTS
The average time from death to refrigeration was 4.2 hours, to enucleation 6.4 hours, and to tissue processing 10.7 hours. RT-PCR gel electrophoresis patterns from retinal tissue had bands of similar intensity at each interval from beta-actin, GAPDH, and RHO. 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 was 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 in which eye bank tissue is used for RNA analysis.
Malik
Khurram J
KJ
University of Minnesota, Department of Ophthalmology, Minneapolis, Minnesota 55455-0501, USA.
Chen
Ci-Di
CD
Olsen
Timothy W
TW
D000199:Actins / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins; D015925:Cryopreservation; D005125:Eye Banks; D015353:Eye Enucleation; D005136:Eye Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D023421:Models, Animal; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011180:Postmortem Changes; D011506:Proteins / Q000235:genetics; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics; D013552:Swine; D013997:Time Factors; D014021:Tissue Preservation; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D000199:Actins; D002352:Carrier Proteins; D005136:Eye Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011506:Proteins; D012313:RNA; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012243:Rhodopsin; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase