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Title: PBMC LNA-array microRNA profiling      
dateReleased:
07-03-2016
description:
T cells were collected from four healthy donors using heparin anti-coagulant, diluted with equal amounts of PBS EDTA, then layered over Lymphoprep and spun to collect PBMCs. CD4+ T cells were purifed using the Miltenyi positive selection magnetic labeling kit. Cells were resuspended in RPMI (Invitrogen) with 10% FCS amd 50 U/ml IL-2 (Immunotools), and 3.5x105 plated out per well in 48 well plates, coated overnight with 2 µg/ml anti-CD3 and either anti-CD28 or anti-CD46. Purity was assessed by flow cytometry and was in all cases ≥ 95%. RNA was purified from a set of unactivated cells before plating, to represent the resting, 0 hr time point samples. For early activation samples, CD28 or CD46 co-stimulated cells were harvested 2 hrs after plating. Then, 36 hours after plating, the remaining CD46-stimulated cells were harvested and labeled for IL-10 or IFN-g secretion using the Miltenyi cytokine detection kits, according to manufacturer’s instructions. Cells were then sorted into separate cytokine-secreting populations (IFN-g+/IL-10–, IFN-g+/IL-10+, and IFN-g–/IL-10+) using a BD biosciences FACS machine.RNA from separate populations was labeled using the Hy3 microRNA (miRNA) Power labeling kit (Exiqon) and hybridised to an LNA (locked nucleic acid)-based miRCURY miRNA microarray slides (Exiqon, version 11) using a Maui hybridisation chamber (BioMicrosystems). Spot signals were acquired with an Agilent array scanner and GenePix Pro 4.1 was used for annotation (mirBase 14.0), signal processing and quantification using default background subtraction.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-MTAB-2658
refinement:
raw
dateSubmitted:
02-22-2010
keywords:
functional genomics
dateModified:
12-28-2016
availability:
available
types:
gene expression
name:
Homo sapiens
name:
stimulus or stress design
ID:
A-GEOD-16352
name:
miRCURY LNA™ microRNA Array V11.0- hsa, mmu & rno
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-2658/E-MTAB-2658.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-2658/E-MTAB-2658.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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