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| Status |
Public on Feb 28, 2022 |
| Title |
treated IL-6 3T3-L1 -rep2 |
| Sample type |
RNA |
| |
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| Source name |
murine 3T3-L1 adipocytes
|
| Organism |
Mus musculus |
| Characteristics |
cell type: 3T3-L1 Mouse Embryonic Fibroblasts
stage of differentiation: on 8th day of adipogenic differentiation
|
| Treatment protocol |
The cells were exposed to IL-6 at concentrations 1 ng/ ml. This concentration reflects post-exercise systemic cytokine levels and increases insulin-stimulated glucose transport in mouse myotubes in vitro. IL-6 was added to the medium at the time of differentiation induction, and after that the medium was changed every 24 h, to support a fresh bolus of the experimental factor. IL-6 (recombinant, expressed in E.coli and suitable for cell culture) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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| Growth protocol |
Adipogenic differentiation in 3T3-L1 cells was carried out free of contamination, using young stock. Cells were seeded and grown until appr. 100% confluence and for further 48 hours, in DMEM with high glucose, supplemented with 10% NBCS (New-Born Calf serum, Invitrogen) and an antibiotic-antimycotic mixture (Life Technologies), in controlled humidified air with 5% CO2, at 37oC. After this time, a differentiating medium was applied, which consisted of DMEM enriched with 10% fetal bovine serum (FBS, Fetal Bovine serum, Invitrogen) and supplemented with prodifferentiative agents (0.5 mmol/l isobutyl-methylxanthine, 1 µmol/l dexamethasone, 2 µmol/l insulin and 2 µmol/l rosiglitazone). After 3 days, this medium was replaced with DMEM supplemented with 10% FBS and insulin (in the concentration indicated above) and the cells were maintained in this medium for further 3 days. The last 2 days of incubation was performed in 10%FBS/DMEM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA, which also included miRNA, was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s procedure. RNA quality was determined using Agilent 2100 Bioanalyzer (USA) and RNA 6000 Nano Kit (Agilent, Germany). All RNA samples taken for microarray analysis were of high quality (RIN was between 9.5 and 10.0).
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNA with miRNA Spike-In (microRNA Spike-In Kit, Agilent Technologies) added were taken for labeling reaction. miRNA was labeled with Cyanine 3-pCp using the Complete Labeling and Hyb Kit (Agilent Technologies) according to the manufacturer’s procedure. The labeled RNA samples were purified on Micro Bio-Spin 6 columns to remove DMSO and unbound Cyanine 3-pCp.
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| |
|
| Hybridization protocol |
Each Cyanine-3-labeled RNA samples were hybridized onto microarray for 20h at 55°C in hybridization rotating oven (Agilent Technologies).
|
| Scan protocol |
Acquisition and analysis of hybridization intensities were performed using Agilent DNA Microarray Scanner G2505C. Images were quantified using Agilent Feature Extraction Software (version 10.7.3.1).
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| Data processing |
The scanned images were analyzed with Feature Extraction Software (version 10.10.1.1) using default parameters (protocol miRNA_1010_Sep10 and Grid: 046066_D_F_20141006) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
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| Submission date |
Mar 02, 2021 |
| Last update date |
Feb 28, 2022 |
| Contact name |
Alicja Majewska |
| E-mail(s) |
alamaj@poczta.onet.pl
|
| Organization name |
Warsaw University of Life Sciences-SGGW Faculty of Veterinary Medicine
|
| Department |
Department of Physiology Sciences
|
| Street address |
Nowoursynowska 159
|
| City |
Warszawa |
| ZIP/Postal code |
02-776 |
| Country |
Poland |
| |
|
| Platform ID |
GPL22861 |
| Series (1) |
| GSE168108 |
Interleukin-6 modifies the expression profile of microRNA in mouse 3T3-L1 adipocytes |
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