|
| Status |
Public on Jun 26, 2015 |
| Title |
PTCs_GW4064_2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse proximal tubule cells, GW4064 (1uM), 24 hours
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57/BJ
gender: male
age: 6 weeks
tissue: primary cultured kidney proximal tubule cells
treatment: GW4064
|
| Treatment protocol |
Experiments were carried out in serum-free DMEM. Cells were incubated by the addition of DMSO or 1μM GW4064 (Sigma-Aldrich (catalog #G5172), St. Louis, MO USA) for 24 hours.
|
| Growth protocol |
Kidney cortices from mice were dissected, sliced, minced and digested in 0.25% trypsin solution (Life Technologies BRL, Grand Island, NY) in a shaking incubator at 37°C for 1 hour. Trypsin was neutralized with growth medium (DMEM and 10% FBS containing 100 U/ml penicillin and 0.1 mg/ml streptomycin). The suspension was pipetted and was passed through a 100-μm cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ). The samples were centrifuged (600 rpm for 5 minutes) to pellet the tubules, washed with 10 ml of medium, centrifuged, and washed twice more. The final pellet, consisting mostly of renal tubules, was resuspended in culture medium (REBM bullet kit, Clonetics), plated onto culture dishes (Nalge Nunc International, Naperville, IL) and incubated at 37°C in a carbon dioxide incubator with medium changes every 2 days until confluent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using Trizol (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-026655 Whole Mouse Genome Microarrays 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), 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 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
Raw intensity data were obtained using Agilent's Feature Extraction Software version 10.7 for array image analysis and the calculation of spot intensity measurements. Data analysis was carried out with R/Bioconductor. The processed intensities and normalized across samples were loaded by using quantile normalization. Differential expression was computed using the limma package. Additional details on the analysis methods can be found at: http://fgcz-bfabric.uzh.ch/wiki/tiki-index.php?page=app.two_groups
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| |
|
| Submission date |
Jun 25, 2015 |
| Last update date |
Jul 27, 2015 |
| Contact name |
Zhibo Gai |
| E-mail(s) |
[email protected]
|
| Phone |
+41 44 556 3143
|
| Organization name |
University Hospital Zurich
|
| Department |
Department of Clinical Pharmacology and Toxicology
|
| Street address |
Wagistrasse 14
|
| City |
Zurich |
| State/province |
Schweiz |
| ZIP/Postal code |
8952 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE70296 |
Effect of GW4064 on primary cultured mouse kidney proximal tubule cells |
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