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Bongsu Kim, Bernard Wen, Rania Kusibati, Tyler Nicholas Heisler-Taylor, Frederick Davidorf, Matthew P Ohr, Michael B Wells, Dino D Klisovoc, John B Allen, Sanjoy K Bhattacharya, Colleen M Cebulla; Optimization of retinal detachment vitreous sample preparation methods for protein analysis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5963.
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© ARVO (1962-2015); The Authors (2016-present)
Vitreous structure can hinder the measurement of consistent protein concentration levels over subsequent assays for research or clinical testing in retinal detachment. The purpose of this study is to compare multiple methods of vitreous sample preparation for the consistency of measured protein levels between multiple assays.
Patient vitreous samples (n=4 /group) were randomly selected from sample collection under an IRB-approved protocol during retinal detachment surgical procedure. BCA assay with 96 well plates was performed for the comparison of five groups of vitreous preparation: 1) vortex (S3200, BioExpress), 2) hand held-homogenization (Disposable pellet mixer and cordless motor, VWR), 3) sonication (Dismembrator-500, Fisher Scientific), 4) the use of lysis buffer (9803S, Cell signaling), and 5) no treatment as a control. Three independent assays were run with each treatment and all 4 subjects. Samples in each assay were run in triplicate accounting for pipetting error. The coefficient of variation (CV %) from the three assays was averaged between the 4 subjects and compared between the 5 methods. Student’s t-test was used for statistical analysis.
The mean CV for the no treatment control was 13.48% ± 9.21. The mean CV of lysis buffer treatment and sonication are significantly lower than that of control (4.34% ± 1.79, P=0.0417 and 4.49% ± 3.14, p=0.0449, respectively). The mean CV of vortex treatment and hand-held homogenization were 17.05% ± 7.73 and 18.43% ± 3.28, respectively. These were not statistically significant compared to control (p=0.3988 and p=0.2464, respectively).
Vitreous sample sonication or lysis buffer extraction significantly improves the consistency of protein measurement of samples. Future study will evaluate the quality of protein detection with ELISA assays after using these methods
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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