June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Optimization of retinal detachment vitreous sample preparation methods for protein analysis
Author Affiliations & Notes
  • Bongsu Kim
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Bernard Wen
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Rania Kusibati
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Tyler Nicholas Heisler-Taylor
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Frederick Davidorf
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Matthew P Ohr
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Michael B Wells
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Dino D Klisovoc
    Midwest Retina, Dublin, Ohio, United States
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • John B Allen
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Sanjoy K Bhattacharya
    Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Colleen M Cebulla
    Havener Eye Institute, Ophthalmology and Visual Science, The Ohio State Universty, Columbus, Ohio, United States
  • Footnotes
    Commercial Relationships   Bongsu Kim, None; Bernard Wen, None; Rania Kusibati, None; Tyler Heisler-Taylor, None; Frederick Davidorf, None; Matthew Ohr, None; Michael Wells, None; Dino Klisovoc, None; John Allen, None; Sanjoy Bhattacharya, None; Colleen Cebulla, None
  • Footnotes
    Support  NIH K08EY022672, P30 CA016058 ( OSU-CCC Nucleic Acids Shared Resource), NCATS KL2TR001068. EY014801 (core grant of BPEI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding institutions. Additional funds were provided by the Ohio Lions Eye Research Foundation, Ophthalmology Fund #313310, and the Patti Blow Fund
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5963. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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).

Conclusions : 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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