|
| Status |
Public on Jan 31, 2018 |
| Title |
infrapopliteal artery 2 |
| Sample type |
RNA |
| |
|
| Source name |
primary SMCs from infrapopliteal artery
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
tissue: infrapopliteal artery
cell type: primary Smooth Muscle Cells (SMCs)
|
| Treatment protocol |
Non atheromatous, healthy arteries were harvested from allografts obtained from deceased donors. Fresh tissue was washed with cold PBS and dissociated with an enzymatic mix including collagenase, elastase and DNase in High glucose DMEM. A first short digestion step permitted endothelial cell isolation. Then mechanical dissociation followed by a longer incubation with the digestion mix allowed SMC isolation. Occlusive debris was removed using a 70 µm mesh strainer. Additional centrifugation removed smaller debris. Cells were plated in gelatin-coated 12 wells plates in complete SMCBM2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Macherey Nagel NucleoSpin columns. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was assessed using an Agilent 2200 TapeStation system.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from100 ng RNA using the Low Input QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labeled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent Hi-RPM hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human Gene Expression microarrays (G4851B) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent). Next, microarrays were washed 30 seconds in acetonitrile and 30 seconds in Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent G2565CA Microarray Scanner using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
H204_6
primary SMCs from infrapopliteal artery rep2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1-107_Sep09 and Grid: 039494_D_F_20120628) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were normalized using R statistical software. Raw values of all probes on all arrays were normalized against a virtual median chip (median raw intensity per row) using a locally weighted scattered plot smoother analysis (LOWESS). Values for replicate probes were averaged. The data were filtered to remove probes with low intensity values.
|
| |
|
| Submission date |
Jul 05, 2016 |
| Last update date |
Jan 31, 2018 |
| Contact name |
Marja Steenman |
| E-mail(s) |
marja.steenman@inserm.fr
|
| Organization name |
L'institut du thorax
|
| Street address |
8 quai Moncousu
|
| City |
Nantes |
| ZIP/Postal code |
44007 |
| Country |
France |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE84012 |
Implication of VSMC heterogeneity among arterial beds in vascular calcification |
|
