|
| Status |
Public on Dec 16, 2017 |
| Title |
Scramble rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
pulmonary arterial smooth muscle cells (hPASMC)
|
| Organism |
Homo sapiens |
| Characteristics |
sirna transfection: Scramble
cell type: pulmonary arterial smooth muscle cells (hPASMC) in P2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 24h following siTgfbr3 transfection, hPASMC were rinsed with PBS and the total RNA was extracted with miRNeasy Mini Kit (Qiagen). 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 |
The total amount of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was hybridized to Agilent-046064 Human miRNA Release 19.0 8x60K Microarray (GPL18402) for 20 hours at 55°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 miRNA_107_Sep09 and Grid: 046064_D_F_20121223) 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 |
GPL18402 |
| Series (1) |
| GSE92554 |
microRNA expression analysis following 24h siRNA knockdown of transforming growth factor β receptor III (TGFBR3) in human pulmonary arterial smooth muscle cells. |
|
