|
| Status |
Public on Nov 23, 2016 |
| Title |
HMB-treated equine satellite cells_Cy5 vs. Equine satellite cells without HMB treatment_Cy3_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HMB-treated equine satellite cells_Cy5
|
| Organism |
Equus caballus |
| Characteristics |
age: 3rd day of differentiation
satellite cell origin: m. semitendinosus
cell type: satellite cell
|
| Treatment protocol |
After the 2nd day of differentiation 50µg β-hydroxy-β-methylbutyrate (HMB) was added and cells were incubated for 24h. Control cells were incubated in differentiation medium.
|
| Growth protocol |
Equine satellite cells were cultured in Primaria culture flask (BD, USA). The growth medium (10%FBS/10%HS/DMEM/AB) was changed every two days. On the 10th day of proliferation cells were transferred to Collagen I Cellware 6-well plate. After obtaining 80% of confluence proliferation medium was replaced by differentiation medium (2%HS/DMEM/AB). FBS - fetal bovine serum, HS - horse serum, DMEM - Dulbecco's Modified Eagle Medium, AB - antibiotics: 1% Penicillin-Streptomycin, 0.5% Amphotericin B.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated according to the protocol supplied with the miRNeasy Mini Kit (Qiagen, USA). RNA quantity was measured spectrophotometrically using NanoDrop (NanoDrop Technologies, USA). The analysis of final RNA quality and integrity was performed with BioAnalyzer 2100 (Agilent Technologies, USA). To ensure optimal data quality only RNA samples with RIN number ≥ 9,2 were included into the analysis.
|
| Label |
Cy5
|
| Label protocol |
Low Input Quick Amp Labeling Kit (Agilent Technologies, USA) was used to amplify and label target RNA to generate complementary RNA (cRNA).
|
| |
|
| Channel 2 |
| Source name |
Equine satellite cells without HMB treatment_Cy3
|
| Organism |
Equus caballus |
| Characteristics |
age: 3rd day of differentiation
satellite cell origin: m. semitendinosus
cell type: satellite cell
|
| Treatment protocol |
After the 2nd day of differentiation 50µg β-hydroxy-β-methylbutyrate (HMB) was added and cells were incubated for 24h. Control cells were incubated in differentiation medium.
|
| Growth protocol |
Equine satellite cells were cultured in Primaria culture flask (BD, USA). The growth medium (10%FBS/10%HS/DMEM/AB) was changed every two days. On the 10th day of proliferation cells were transferred to Collagen I Cellware 6-well plate. After obtaining 80% of confluence proliferation medium was replaced by differentiation medium (2%HS/DMEM/AB). FBS - fetal bovine serum, HS - horse serum, DMEM - Dulbecco's Modified Eagle Medium, AB - antibiotics: 1% Penicillin-Streptomycin, 0.5% Amphotericin B.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated according to the protocol supplied with the miRNeasy Mini Kit (Qiagen, USA). RNA quantity was measured spectrophotometrically using NanoDrop (NanoDrop Technologies, USA). The analysis of final RNA quality and integrity was performed with BioAnalyzer 2100 (Agilent Technologies, USA). To ensure optimal data quality only RNA samples with RIN number ≥ 9,2 were included into the analysis.
|
| Label |
Cy3
|
| Label protocol |
Low Input Quick Amp Labeling Kit (Agilent Technologies, USA) was used to amplify and label target RNA to generate complementary RNA (cRNA).
|
| |
|
| |
| Hybridization protocol |
On each two-colour microarray 825 ng of cRNA from GO exposed cells (Cy5) and 825 ng of cRNA from control cells (Cy3) were hybridized using Gene Expression Hybridization Kit (Agilent Technologies, USA) according to the manufacturers protocol. RNA Spike In Kit (Agilent Technologies, USA) was used as an internal control.
|
| Scan protocol |
Acquisition and analysis of hybridization intensities were performed using the Agilent DNA microarray scanner and Agilent Feature Extraction software 10.7.3.1. according to the standard manufacturer’s procedures and LOWESS normalization.
|
| Description |
Biological replicate 1 of 4. GO-treated equine satellite cells_Cy5 vs. Equine satellite cells without HMB treatment_Cy3_rep1
|
| Data processing |
The statistical analysis was performed using Gene Spring 13 software default protocol (Agilent Technologies, USA). The statistical significance of the differences was evaluated using student test (p<0.05) and fold change FC>1.3. A multiple testing correction was performed using Benjamini and Hochberg False Discovery Rate (FDR) ≤0.05.
|
| |
|
| Submission date |
Oct 29, 2015 |
| Last update date |
Nov 23, 2016 |
| Contact name |
Tomasz Sadkowski |
| E-mail(s) |
tomasz_sadkowski@sggw.pl
|
| Phone |
+48225936263
|
| Fax |
+48228472452
|
| URL |
http://sadkowski.info
|
| Organization name |
Warsaw University of Life Sciences - SGGW
|
| Department |
Department of Physiological Sciences
|
| Street address |
Nowoursynowska 159
|
| City |
Warsaw |
| ZIP/Postal code |
02-776 |
| Country |
Poland |
| |
|
| Platform ID |
GPL15189 |
| Series (1) |
| GSE74495 |
Expression data from differentiating primary culture of equine satellite cells (ESC) β-hydroxy-β-methylbutyrate (HMB) |
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