Abstract
Abstract: :
Purpose: cDNA libraries have been produced from the whole mouse eye and the mouse retina, no well characterized library from the RPE or choroid of the mouse has been reported. We developed optimal RNA preparation procedures in order to construct an RPE/choroid library for use in studies of this tissue. Methods: Eyes from 100 6 week C57/Bl6 mice were dissected and processed in groups of 10. RNAlater was added to individual eye cups after removal of the neural retina. The RPE/choroid was then scraped from the scleral wall and processed by sequential trituration through a 21 guage needle, QIAshredder preparation, and then RNeasy column preparation of total RNA. Isolated total RNA was checked for integrity by electrophoresis and then poly A RNA was prepared from the pooled total sample and cloned into the Sport1 vector system. Results: After plating, 3,500 individual cDNA clones were picked and sequence from the 5’ end. Sequences were grouped into clusters and identified using informatics tools on the NEIBank. Of the 3207 clusters identified, most (80%) represented single clones. The most abundant sequences identified were genes known to be expressed in the RPE. The entire vitamin A pathway, for example, was found in the library as well as many RPE- specific genes previously reported. RPE65 and opsin was investigated by in situ hybridization to examine expression patterns in the posterior pole. Conclusions: The total RNA isolated from the RPE/choroid of 1 mouse eye (0.5 micrograms) was only 10% of the total RNA of 1 retina (5 micrograms). The resulting library from this sample was very flat, with only 20 % of the sequences appearing more than once after sequences were clustered. About 10% sequences were identified which had no match to other published sequences, but none of these appeared in clusters with 2 or more clones. It is likely that deeper sequencing will substantially expand the library.
Keywords: choroid • aging • retinal pigment epithelium