Jun 17, 2020
MicroRNA Affymetrix microarray probe labeling from ocular fluids
- 1University of California, Davis
External link: https://doi.org/10.1371/journal.pone.0235541
Protocol Citation: Zeljka Smit-Mcbride 2020. MicroRNA Affymetrix microarray probe labeling from ocular fluids. protocols.io https://dx.doi.org/10.17504/protocols.io.bc7pizmn
Manuscript citation:
Smit-McBride Z, Nguyen AT, Yu AK, Modjtahedi SP, Hunter AA, Rashid S, Moisseiev E, Morse LS (2020) Unique molecular signatures of microRNAs in ocular fluids and plasma in diabetic retinopathy. PLoS ONE 15(7): e0235541. doi: 10.1371/journal.pone.0235541
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 04, 2020
Last Modified: June 17, 2020
Protocol Integer ID: 33743
Abstract
Sample collection, isolation of miRNAs, and quality control
Aqueous and vitreous humor, as well as plasma samples, have been collected from Diabetes mellitus (DM) and control patients during the standard-of-the-care eye surgery. Samples of 100-200ul were collected, aliquoted in 100ul aliquots, frozen on dry ice immediately and stored at -80oC. RNA was isolated using Exiqons’ modification of Qiagen’s microRNeasy kit. Quantification and quality check of isolated miRNAs was performed on BioAnalyzer (Agilent) with Small RNA microfluidics Chips.
Affymetrix microarray probe labeling
Probe labeling was done from 10 ng of aqueous, 20ng of vitreous and 20ng of plasma miRNA samples from a total of 24 aqueous, 24 vitreous and 25 plasma samples using FlashTag (Affymetrix) procedure. Labeled probes were hybridized to Affymetrix miRNA Array 3.0 in UC Davis Genome Center Microarray Core Facility using the standard procedure (Affymetrix, Santa Clara, CA). The total data set included 73 Affymetrix miRNA 3.0 microarrays. Upon scanning of the hybridized and washed Chips, data was obtained in a set of Affymetrix data files (.cel, .arr, .jpg).