|
| Status |
Public on Jan 07, 2014 |
| Title |
9-RT-314 |
| Sample type |
RNA |
| |
|
| Source name |
Cardiac non-proliferating myofibroblasts
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar rats
gender: male
weight: 200-250 g
tissue: left ventricular cardiac tissue
cells: cardiac fibroblasts
|
| Biomaterial provider |
Laboratory of Hypertension, KU Leuven
|
| Treatment protocol |
Treatment with TGF-β1
|
| Growth protocol |
second passaged Fb were seeded at a density of 2600 cells/cm2 and cultured in DMEM medium with 10% fetal bovine serum and 1% of penicillin/streptomycin. To test fibroblast differentiation pathways we used SD-208 (3 µmol/L; Sigma-Aldrich, Belgium), a specific TGF-β receptor I kinase inhibitor during 4 days and recombinant human TGF-β1 (400 pmol/L; PeproTech, USA) during 6 days in culture. Spontaneous differentiation in standard culture medium was performed during 4 days in culture. All the experiments comparing cell differentiation were a paired study design, i.e. the different treatments were applied to cultures from the same heart. Experiments were repeated in 4 animals.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from p-MyoFb, SD-208 treated and TGF-β1 treated cell cultures using TRIzol reagent (Invitrogen, Belgium) and purified on RNeasy Mini Kit columns (Qiagen). 0.5 µg of total RNA was used for cDNA synthesis with the SuperScript VILO cDNA synthesis kit (Invitrogen, Belgium). The cDNA was diluted 50-fold. qRT-PCR was performed on a 7500 Fast Real-Time PCR machine using the SYBRGreen master mix (Applied Biosystems, Belgium). The relative gene expression was calculated by comparing cycle times for target PCR using the following equation: relative gene expression = 2−(ΔCtsample—ΔCtcontrol.
|
| Label |
biotin
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit.
|
| |
|
| Hybridization protocol |
A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Rat Gene 2.0 ST Arrays followed by staining and washing in a GeneChip® fluidics station 450 (Affymetrix) according to the manufacturer’s procedures.
|
| Scan protocol |
To assess the raw probe signal intensities, chips were scanned using a GeneChip® scanner 3000 (Affymetrix).
|
| Description |
Abrreviation: non-p-MyoFb
|
| Data processing |
The RMA expression values were computed with the xps package (version 1.20.2) of Bioconductor.
|
| |
|
| Submission date |
Oct 29, 2013 |
| Last update date |
Jan 07, 2014 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL17117 |
| Series (1) |
| GSE51824 |
Reversible and irreversible differentiation of cardiac fibroblasts |
|
