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Justine J Cunningham, Maria Burton, Gerry Rodrigues, Thomas C Hohman; Optimized Dual Protein/miRNA Method to Quantitate Clinical Biomarkers of Ocular Disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2299.
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© ARVO (1962-2015); The Authors (2016-present)
A key challenge to defining supportive clinical biomarkers that can be correlated with therapeutic benefit in patients undergoing treatment for ocular disease, is the limited volume of clinical ocular samples (≤25-50µL’s). Our goal was to develop and optimize a dual protein/RNA extraction method to identify biomarkers of ocular disease that might be correlated with target engagement in the retina/choroid. Advantages of miRNAs are that they: 1) inform about regulatory networks rather than a single target, and 2) require a very small sample (≤5µL).
Six pairs of human eyes (NDRI, NY) were dissected and tissues processed following either; 1) an RNA-only or 2) a dual protein/RNA extraction method. RNA Only: tissues were placed directly in the RNA denaturing buffer and processed according to manufacturer’s instructions. Dual RNA/protein: tissues were first homogenized in the buffers compatible for downstream protein analysis. A portion of the homogenate was set aside for RNA extraction. Four different RNA isolation kits for small RNAs – PARIS, mirVana, miRVana PARIS (Ambion) and miRNeasy (Qiagen) kits – were tested and compared for optimized isolation and total RNA quality based on A260/280; A230/280 ratios; and RIN values (Bionanalyzer, Agilent Technologies). Total RNA preparations enriched for small RNAs were sent to Qiagen for a miRNome analysis using qRT-PCR SYBR green technology. This analysis was able to detect most miRNAs known to date (2,034 miRNAs).
By using the same homogenization buffer (TER-I) used for downstream protein assays we were able to maintain optimal conditions for protein analyses while extracting high quality RNA for miRNA profiling. We also obtained higher total RNA yield and ‘purity’ from both retina and choroid when using Ambion’s mirVana kit versus Qiagen’s miRNeasy kit. All RNA samples submitted to Qiagen for quality control (QC) testing met the criteria for miRNA profiling. We determined the expression levels (CT) of 2,032 miRNAs in retina/choroid tissues.
We developed and optimized a method that would allow us to determine expression levels of miRNAs in normal versus diseased ocular tissues e.g. AMD. Together with determination of target protein levels, this analysis will enhance our understanding of miRNA changes in ocular disease and aid us in identifying potential biomarkers to support target engagement.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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