|
| Status |
Public on Oct 25, 2016 |
| Title |
primary lung fibroblasts_ scr. RNA_rep.4 |
| Sample type |
RNA |
| |
|
| Source name |
primary lung fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
tissue: primary lung fibroblasts
transfection: scr. siRNA
|
| Treatment protocol |
Following the isolation protocol, primary murine lung fibroblasts were cultured and passaged at least 3 times before the experiment. Following seeding in 6-well plate, cells were transfected with either control (scrambled) or sequence-specific siRNA against Lysyl oxidase (Lox), Lysyl oxidase-like (Loxl1) or Lysyl oxidase-like 2 (loxl2) genes. Cells were harvested 48 hours after the transfection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from the cells was isolated using a peqlab total RNA extraction kit.
|
| 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-028005 SurePrint G3 Mouse GE 8x60K Microarray (GPL10787) 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%).
|
| Description |
Gene expression 48 hours after scr siRNA transfection
|
| 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: 028005_D_F_20121126 for RS-284 samples or Grid: : 028005_D_F_ 20120201 for RS-266 samples ) 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 of either one of the experiments RS-266 or RS-284, than they were excluded.
|
| |
|
| Submission date |
Oct 24, 2016 |
| Last update date |
Oct 25, 2016 |
| 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 |
GPL10787 |
| Series (1) |
| GSE89121 |
Exogenous expression of Lox, Loxl1 and Loxl2 regulate gene expression in primary murine lung fibroblasts. |
|
