|
| Status |
Public on Dec 16, 2017 |
| Title |
siRNA Tgfbr3 rep4 |
| Sample type |
RNA |
| |
|
| Source name |
pulmonary arterial endothelial cells (hPASMC)
|
| Organism |
Homo sapiens |
| Characteristics |
sirna transfection: siRNA Tgfbr3
cell type: pulmonary arterial endothelial cells (hPAEC) in P2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 24h following transfection hPAEC were rinsed with PBS and the total RNA was extracted with peqGOLD TOTAL RNA Kit (VWR). RNA was frozen in liquid nitrogen and stored in -80°C prior to the shipping.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low Input Quick-Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-028004 SurePrint G3 Human GE 8x60K Microarray (GPL14550) for 18 hours at 65°C in a rotating 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), then dried immediately by brief centrifugation
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20110819) to obtain background subtracted and spatially detrended Processed Signal intensities. Features which were not positive and significant, not above background or were population outliers were flagged as “Compromised”. If features were flagged “Compromised” in all samples, than they were excluded.
|
| |
|
| Submission date |
Dec 19, 2016 |
| Last update date |
Dec 16, 2017 |
| Contact name |
Rory Morty |
| Organization name |
MPI für Herz- und Lungenforschung
|
| Department |
W.G. Kerckhoff-Institut
|
| Street address |
Parkstr. 1
|
| City |
Bad Nauheim |
| ZIP/Postal code |
61231 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE92553 |
Gene expression analysis following 24h siRNA-mediated knockdown of transforming growth factor β receptor III (TGFBR3) in human pulmonary arterial endothelial cells (hPAEC). |
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