|
| Status |
Public on May 01, 2014 |
| Title |
BXPC3_DDR1shRNA_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
BXPC3 DDR1 shRNA, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Pancreatic cancer cell line
|
| Growth protocol |
BXPC3 cell lines were plated at 2 million cells/10 cm dish in triplicate and grown RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated for 72 hours at 37 ºC in a humidified incubator with 5% CO2 prior to harvest for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cells with TRIzol Reagent (Life Technologies, Carlsbad, USA) according to manufacturer protocol. RNA was digested with DNase I-Amp Grade (Life Technologies) on-column and purified using an RNeasy Mini kit (QIAGEN, Valencia, USA) according to manufacturer protocols. RNA quality and quantification were performed by analyzing RNA in RNA Nano 6000 chips in a Bioanalyzer2100 following manufacturer protocol (Agilent Technologies, Santa Clara, USA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine 3-CTP labeled cRNA target was prepared using One-Color Low Input Quick Amp kit and One Color Spike-In kit (Agilent Technologies) using 200 ng input RNA (RIN>8.0) according to manufacturer protocols. Labeled cRNA was purified using RNeasy Mini kit (QIAGEN) according to manufacturer recommendation. Labeled cRNA was quantified on a NanoDrop ND-1000 UV Spectrophotometer using Microarray/RNA-40 measurement. Specific activity of Cyanine 3 was calculated in pmol /µg with a minimum threshold of 15 pmol/µg.
|
| |
|
| Hybridization protocol |
1650 ng of labeled cRNA was fragmented by incubation with 10 µl of 10X Blocking Reagent and 25X Fragmentation Buffer in a 50 µl reaction volume for 30 minutes at 65 °C. 50 µl of 2X GE Hybridization Buffer HI-RPM was added to fragmented cRNA and hybridization mixes then placed onto Agilent Human GE 4X44K v2 microarrays. Hybridization was done for 17 hours at 65 °C by rotating at 10 rpm. Microarray slides were washed in GE Wash Buffer 1 for 2 minutes and pre-warmed GE Wash Buffer 2 for 2 minutes at room temperature.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 09, 2012 |
| Last update date |
May 01, 2014 |
| Contact name |
Mike Mattie |
| E-mail(s) |
mmattie@agensys.com
|
| Organization name |
Agensys Inc
|
| Department |
Applied Molecular Biology
|
| Street address |
1800 Stewart Street
|
| City |
Santa Monica |
| State/province |
CA |
| ZIP/Postal code |
90404 |
| Country |
USA |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE39207 |
DDR1 knockdown in BXPC3 cell line |
|
