|
| Status |
Public on May 11, 2016 |
| Title |
Dex and COS conditions rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C2C12
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Myotubes
treatment: DEX+COS culture conditions day 5-8 post differentiation: 4% O2, low-frequency (1Hz/0.1Hz for 18h/6h respectively) biphasic 5ms square-wave electrical stimulation, and daily TNF-α (20 pg/ml; R&D systems) and IL-6 (100 pg/ml; R&D systems) + daily Dexamethasone (1µM)
|
| Treatment protocol |
All treatments started day 5 post differentiation including transfer to new culture conditions (COS) and/or treatment with additional soluble agents. All media and treatments were changed every 24hrs prior to harvesting cells (D5-D7).
|
| Growth protocol |
C2C12 myoblasts (ATCC) less than 10 passages grown in high-glucose DMEM (Gibco) with 10% FBS (invitrogen) seeded on 0.15% gelatin-coated 6-well plates at at 2.2 x 10^6 cells per well, grown to 80% confluence and differentiated in medium containing 10% horse serum. All initial cultures were performed in standard tissue culture conditions (21% O2, 5% CO2, humidified incubator at 37C)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the miRNeasy mini protocol (Qiagen) as per the manufacturer's instructions
|
| Label |
Hy3
|
| Label protocol |
2µg total RNA from each experimental sample was covalently 3’-labeled with a single Hy3 (test) or Hy5 (reference) fluorophore per molecule using the Hi-Power Labeling kit (Exiqon, Woburn MA)
|
| |
|
| Channel 2 |
| Source name |
C2C12
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Myotubes
treatment: COS culture conditions day 5-8 post differentiation: 4% O2, low-frequency (1Hz/0.1Hz for 18h/6h respectively) biphasic 5ms square-wave electrical stimulation, and daily TNF-α (20 pg/ml; R&D systems) and IL-6 (100 pg/ml; R&D systems)
|
| Treatment protocol |
All treatments started day 5 post differentiation including transfer to new culture conditions (COS) and/or treatment with additional soluble agents. All media and treatments were changed every 24hrs prior to harvesting cells (D5-D7).
|
| Growth protocol |
C2C12 myoblasts (ATCC) less than 10 passages grown in high-glucose DMEM (Gibco) with 10% FBS (invitrogen) seeded on 0.15% gelatin-coated 6-well plates at at 2.2 x 10^6 cells per well, grown to 80% confluence and differentiated in medium containing 10% horse serum. All initial cultures were performed in standard tissue culture conditions (21% O2, 5% CO2, humidified incubator at 37C)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the miRNeasy mini protocol (Qiagen) as per the manufacturer's instructions
|
| Label |
Hy5
|
| Label protocol |
2µg total RNA from each experimental sample was covalently 3’-labeled with a single Hy3 (test) or Hy5 (reference) fluorophore per molecule using the Hi-Power Labeling kit (Exiqon, Woburn MA)
|
| |
|
| |
| Hybridization protocol |
Performed on fully-automated enclosed hybridisation station (Tecan HS400Pro). Samples were injected manually then co-hybridised (56 degrees C x 16 hrs) then washed and dried automatically prior to scanning
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
|
| Description |
Biological replicate 2 of 2: Dex treatment under COS conditions vs COS conditions
|
| Data processing |
Two-colour images were analysed using Spot software (CSIRO, 2007) and mean signal intensity for each spot was determined after background correction and Loess print-tip (within-array) normalisation of the log2 ratio (FlexArray, Genome Quebec, McGill University).
Normalized log2 ratio (Hy3/Hy5)
|
| |
|
| Submission date |
May 10, 2016 |
| Last update date |
May 11, 2016 |
| Contact name |
R. Thomas Jagoe |
| E-mail(s) |
thomas.jagoe@mcgill.ca
|
| Phone |
15143408222
|
| Organization name |
McGill University
|
| Department |
Medicine
|
| Street address |
Jewish General Hospital, 3755 Cote Ste Catherine
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3T 1E2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL21850 |
| Series (1) |
| GSE81138 |
miRNA profiling of mature C2C12 myotubes using modified (COS) vs standard (STD) tissue culture conditions |
|
