Peripheral blood mononuclear cells (PBMCs) were separated from the 20 ml blood samples obtained from the patients in acute, chronic and healthy control groups via intensity gradient centrifuge method with Ficoll (Biochrom, Germany). PBMC fraction of patient whole blood was stained using CD4-FITC, CD8 PE, CD3 PerCP (Beckton Dickinson (BD), Biosciences, USA) antibodies to distinguish the CD8+ T cells for FACS. Antibodies were supplied by BD Biosciences, Inc. (USA) and used at the minimal saturating concentration, determined by titration prior to the experiment. PBMCs were stained with the antibody combination for 20 minutes at room temperature, washed twice and resuspended in PBS supplemented with 1% FCS. CD8+ T cells were sorted on FACSAria (BD Biosciences cell-sorter, USA) and kept at 4°C throughout sorting. Live PBMCs were gated based on forward versus scatter profiles, followed by CD3+ based gating for T lymphocyte identification. The CD3+ positive population was subdivided by CD8/CD4 expression and used for the identification of CD8+ (CD4–CD8+) T cells. Purity was determined for CD8+ T cells isolated by FACS for CD8+ T cells (99,8 % purity) Sorted cells were immediately centrifuged and pellets were resuspended in TriPure Isolation Reagent (Roche, Germany) at -80°C until use. Cells were stored with TriPure Isolation Reagent (Roche, Germany) at -80°C until use. miRNeasy Kit (Qiagen, Germany) was used in accordance with the manufacturer protocol for the total RNA isolation from the isolated CD8+ T cells. The measurements of concentrations and purities were carried out using the Nanodrop (Thermo Scientific, USA) equipment. RNA qualities were determined in Agilent Bioanalyzer (Agilent, UK) device using Agilent RNA 6000 Nano Kit.
Label
Cy3
Label protocol
MicroRNAs from RNA samples were marked with Cy3 fluorescent dye while transforming them into cDNA using 100ng RNA Agilent miRNA labeling Kit (Agilent, UK)
Hybridization protocol
Spike Kit (Agilent, UK) and Agilent microRNA Hybridization Kit (Agilent, UK) were used to hybridize into Human miRNA Microarray, Release 19.0, 8x60K(v19) microarray slides (Agilent, UK)
Scan protocol
Human miRNA Microarray, Release 19.0, 8x60K(v19) microarray slides (Agilent, UK) scanned using Nimblegen MS200 array scanner.
Description
microRNA expression in chronic brucella infection_sample 3
Data processing
The TIFF image files obtained were processed using Agilent Feature Extraction software to extract raw data and obtain QC reports.
MICRORNA EXPRESSİON PATTERNS OF CD8+ T CELLS IN ACUTE AND CHRONIC BRUCELLOSIS
Data table header descriptions
ID_REF
VALUE
The data of the samples that pass QC parameters were subject to quantile normalization and analyzed using GeneSpring GX (Agilent, UK) 13.0 software after which microRNA’s with P values of >0,05 and Fold change values of >2 and <-2 were determined to be statistically significant.
