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Sample GSM1435491 Query DataSets for GSM1435491
Status Public on Jul 12, 2014
Title IL-13 treated macrophages 2
Sample type RNA
 
Source name primary human monocyte-derived macrophages with IL-13 treatment for 48hr-2
Organism Homo sapiens
Characteristics tissue: buffy coats of peripheral blood
cell type: macrophage
donors: healthy donor 2
Treatment protocol Afterward, macrophages were incubated with or without 20ng/ml IL-4 or 20ng/ml IL-13(PeproTech, Rocky Hill, NJ) for 48 hr.
Growth protocol Peripheral blood monocytes from healthy donors were isolated by Ficoll density gradient centrifugation as previously described. Macrophages were obtained by culturing monocytes in DMEM medium supplemented with 10% heat inactivated human AB serum for 6 days.
Extracted molecule total RNA
Extraction protocol Total RNA from human primary macrophages treated with or without 20 ng/ml IL-4 or 20ng/ml IL-13 for 48 hr was isolated with a mirVana miRNA Isolation kit (Ambion, Foster, CA).
Label Hy3
Label protocol After RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3TM fluorescent label, using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
 
Hybridization protocol After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual. The total 25 μL mixture from Hy3TM-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm.
Scan protocol Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
Description 3'-end-labeled with Hy3TM fluorescent label
miRNAs expression of primary human monocyte-derived macrophages with IL-13 treatment for 48hr-2
Data processing Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor.
Expressed data were normalized using the Median normalization. After normalization, differentially expressed miRNAs were identified through Fold Change filtering.
 
Submission date Jul 11, 2014
Last update date Jul 12, 2014
Contact name Shicheng Su
E-mail(s) seasonso@163.com
Phone 84-02-87332022
Organization name Sun-Yat-Sen Memorial Hospital
Street address 107 Yanjiang West Road
City Guangzhou
ZIP/Postal code 510120
Country China
 
Platform ID GPL17107
Series (2)
GSE59348 M2 cytokines alter the miRNA expression profile in macrophages
GSE59349 The differential miRNA expression induces M2 macrophage polarization and mediates their profibrogenic effects

Supplementary file Size Download File type/resource
GSM1435491_IL-13_treated_macrophages_2.txt.gz 42.0 Kb (ftp)(http) TXT
Processed data are available on Series record

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