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Katarzyna Goode, Rebecca J Kapphahn, Sandra Rocio Montezuma, Deborah A Ferrington; Gene expression analysis from human donor retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4990.
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Identification of pathologic changes in gene expression requires good quality mRNA from both healthy and diseased tissue. The purpose of this research is to optimize methods for retrieving high quality, tissue-specific mRNA for investigating diseases of the eye.
Donor eyes obtained by the Minnesota Lions Eye Bank were acquired with consent of the donor or family for medical research. Donor eyes were enucleated 4 hrs or less from death, fundus imaging and harvesting of tissue was performed within 6 hrs post-mortem. The Minnesota Grading System was used to assign the level of AMD based on fundus images. Following harvest, retinal pigment epithelial (RPE) cells from one eye were placed directly into RNAprotect cell reagent (Qiagen) and stored at -20 degrees until RNA isolation. Total RNA extraction was performed using RNeasy Micro Kit (Qiagen). RNA quality was evaluated at 1, 4, 8, and 14 weeks post isolation using an Agilent Bioanalyzer 2100 (Biomedical Genomics Center, U MN). Complementary DNA was transcribed from mRNA and used to measure the expression of prototypic RPE genes and potential contaminants from endothelial, mast, immune and photoreceptor cells by qPCR using an iQ5 Multicolor Real-Time PCR Detection System (BioRad). PCR products were verified by the appearance of a single peak on melt curves and by a single band migrating at the correct size on a 2% agarose gel.
Samples used for analysis included donors without AMD (male, 87yo, female 56yo) and with advanced AMD (female, 87yo). Total yield of RNA was 7.92 μg for AMD4 donor and 12.44 ± 0.9 μg for AMD1 donors. RNA integrity was excellent. Average RNA Integrity Number (RIN) was 7.6 ± 0.7 for 3 separate donor samples. RIN values were very stable; average change over time (1-14 weeks post isolation) was 6% for each sample. PCR products were obtained for PEDF, RPE65, Best1, CRALBP and reference genes GAPDH, Cyclophillin.
With a short time from death to preservation of RNA using RNAprotect reagent, high quality RNA can be produced from RPE cells harvested from human donor eyes. Samples processed in RNAprotect yielded high quality RNA that was stable over at least 14 weeks post isolation, thus extending the time for generating cDNA. The cDNA from RPE isolated from human donor eyes can be used for comparing gene expression in donors with or without disease, or with cultured RPE to help validate their authenticity.
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