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Rosa M Corrales, Ronil Patel, William Stringer, Jeremy Brace, Barry Butler; Expression of Housekeeping gene, Hypoxanthine Phosphoribosyltransferase-1 (HPRT-1) in Conjunctival Epithelial Cells Under Various Storage Conditions. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2770.
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© ARVO (1962-2015); The Authors (2016-present)
Conjunctival epithelial (CE) cell samples can be used in clinical trials to determine the number of gene expression of certain inflammatory mediators. The expression of these inflammatory mediators may be useful as biomarkers of ocular surface diseases. The purpose of this study was to validate the stability of RNA from CE cells in a defined storage medium (SM) under various storage conditions. The storage conditions simulated the temperatures to which a CE sample might get exposed after collection at a clinical trial site and during shipping of the sample to a bioanalytical lab for processing to determine the biomarker levels using polymerase chain reaction (PCR) analysis. The level of extracted RNA in nanograms, and the number of gene transcripts of the housekeeping gene HPRT-1 were used to determine acceptability of a specific sample that has undergone various storage conditions.
Eighteen CE samples were collected using the Eyeprim™ device from healthy volunteers and stored in SM. To simulate refrigerated storage at the clinical site, all samples were stored at 4°C for 20 days. To simulate various shipping conditions and excursions, duplicate samples were stored under stress (42°C) and normal conditions (4°C-15°C), with or without a cold pack shipping unit, for 24 hours. Temperature was continuously recorded using a temperature logger. RNA was isolated after the maximum allowable time, the concentration was measured and stored at -20°C for 9 days. After storage, RNA was re-measured and cDNA synthesis was performed. The number of gene transcripts of HPRT-1 was quantified using a PCR absolute quantitation method.
The change in RNA concentrations measured after 9 days of storage at -20°C was 0.03%, the average RNA concentration before and after 9 days of storage was 1176±559 ng and 1209±602 ng respectively and was not temperature dependent. The average number of gene transcripts for HPRT-1 was 1248±430 and was not temperature dependent.
Sample storage at various temperature conditions and duration of storage has no effect on the expression levels of housekeeping gene HPRT-1 in CE cells. This validates the sample storage and shipping procedures of the conditions tested for biomarker testing in clinical trials.
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