|
| Status |
Public on Nov 13, 2008 |
| Title |
Liver_PCN_PXR_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver, PCN treated
|
| Organism |
Mus musculus |
| Characteristics |
Genetic background: PXR deficient
Strain: C57BL/6J
Gender: male
Age: 9-12 weeks
Tissue: liver
Treatment: 40 mg/kg PCN
|
| Biomaterial provider |
Viola Tamasi
|
| Treatment protocol |
Animals were injected i.p. 40mg/kg PCN (5% DMSO in corn oil). After 12h they were sacrificed.
|
| Growth protocol |
Animals had free acess to food (standard laboratory chow) and drinking water. They were maintained in 12-hour light/12-hour dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer protocol and purified by RNeasy Mini Kit (Qiagen). Spectrofotometer (Nanodrop) was used to monitor the quality of RNA. RNAs from 6 replicates were pooled and used for hybridization.
|
| Label |
Cy5
|
| Label protocol |
Firefly luciferase (Promega) was added as an universal reference for microarray experiment. 10 μg of total RNA was used for labeling using Agilent Fluorescent Direct Label Kit (Agilent) according to manufacturer protocol.
|
| |
|
| Channel 2 |
| Source name |
Liver, untreated PXR pool
|
| Organism |
Mus musculus |
| Characteristics |
Genetic background: PXR deficient
Strain: C57BL/6J
Gender: male
Age: 9-12 weeks
Tissue: liver
Treatment: 5% DMSO in corn oil
|
| Biomaterial provider |
Viola Tamasi
|
| Treatment protocol |
Animals were injected i.p. wit vehicle (5% DMSO in corn oil). After 12h they were sacrificed.
|
| Growth protocol |
Animals had free acess to food (standard laboratory chow) and drinking water. They were maintained in 12-hour light/12-hour dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer protocol and purified by RNeasy Mini Kit (Qiagen). Spectrofotometer (Nanodrop) was used to monitor the quality of RNA. RNAs from 6 replicates were pooled and used for hybridization.
|
| Label |
Cy3
|
| Label protocol |
Firefly luciferase (Promega) was added as an universal reference for microarray experiment. 10 μg of total RNA was used for labeling using Agilent Fluorescent Direct Label Kit (Agilent) according to manufacturer protocol.
|
| |
|
| |
| Hybridization protocol |
Steroltalk V2 slides were washed before hybridization at room temperature 5 min in 2xSSC and 0.1%SDS, 3 min in 0.2xSSC and 2 min in MilliQ water and dried by centrifugation. Samples were denatured and hybridized in 3xSSC and 0.2% SDS hybridization buffer at 65˚C for 16 hours using humidified hybridization chambers (HybChambers, GeneMachines). After hybridizations slides were washed at room temperature for 5 min in 2xSSC and 0.5% SDS, followed by 5 min in 0.5xSSC, then 5 min in 0.05xSSC and dried by centrifugation.
|
| Scan protocol |
Slides were scanned with Affimetrix 428 Array Scanner using Software Jaguar 2.0. Images were analyzed Gene Pix Pro 6.0 (Molecular Devices Corporation). The median feature and local background intensities were extracted together with the estimates of their standard deviation.
|
| Description |
No additional data.
|
| Data processing |
Features were filtered out according to the following criteria: coefficient of variation (CV, ratio between standard deviation of the background and the median feature intensity) above 0.5 and foreground to background ratio above 1.5. Saturated features were removed. Log2 ratios were normalized using LOESS fit to spike in control RNAs and housekeeping controls. Filtration and normalization was done in Orange software. Normalized log ratios can be compared across samples.
|
| |
|
| Submission date |
Aug 25, 2008 |
| Last update date |
Nov 12, 2008 |
| Contact name |
Peter Juvan |
| Phone |
+386 1 543 7595
|
| Organization name |
Faculty of Medicine, University of Ljubljana
|
| Department |
Institute of Biochemistry
|
| Lab |
Center for Functional Genomics and Bio-Chips
|
| Street address |
Zaloska 4
|
| City |
Ljubljana |
| ZIP/Postal code |
SI-1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL7190 |
| Series (1) |
| GSE12537 |
Effect of PCN on CAR and PXR regulated genes involved in circadian rhythm, drug metabolism and cholesterol homeostasis |
|
