|
| Status |
Public on Jan 29, 2015 |
| Title |
Liver 020 (50mg.kg GSK932121) |
| Sample type |
RNA |
| |
|
| Source name |
Liver Median Lobe - Excised Fresh and Snap Frozen in Liquid Nitrogen
|
| Organism |
Rattus norvegicus |
| Characteristics |
Sex: Female
strain: Crl:CD(SD)
age: 12 weeks
tissue type: Liver
|
| Treatment protocol |
Rats were divided into 2 groups of 12 animals, each of which received a single intraperitoneal (IP) dose of either vehicle or GSK932121A. All doses and concentrations expressed are in terms of the parent compound. A nanomilled and spray-dried formulation of GSK932121A, containing mannitol, hydroxypropylmethylcellulose (Pharmacoat 603) and sodium dodecyl sulphate was suspended in sterile water for IP administration. A dose of 50 mg/kg was selected based on data generated in previous GSK932121A single-dose tolerability studies (known to cause acute toxicity in rats). A single dose of either 50 mg/kg GSK932121A or vehicle (described above minus test article) was administered to female rats (12/group) at a dose volume of 10 mL/kg.
|
| Growth protocol |
Twenty-four female Crl:CD(SD) rats were obtained from Charles River UK (Margate, Kent), and were housed under controlled environmental conditions (19-23°C, 55% ± 10% relative humidity, 12h dark-light cycles), with free access to both water and 5LF2 EURodent Diet (Purina Mills International, St Louis, MO, USA). All animals were implanted with a Plexx™ microchip (Plexx B.V., Elst, The Netherlands) on arrival for the purposes of identification and body temperature measurement. Animals were acclimatised for at least 12 days prior to dose administration, and were 10 to 14 weeks old on the day of dosing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from snap frozen median lobe of liver using the SV Total RNA isolation system (Promega, Madison, WI, USA) according to manufacturers recommendations.
|
| Label |
Biotin
|
| Label protocol |
Fragmented single-stranded DNA was labelled by terminal deoxynucleotidyl transferase (TdT) with Biotin Allonamide Triphosphate using the Affymetrix WT Terminal Labelling and Hybridization Kit (#901524, Affymetrix, Santa Clara, CA, USA)
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| |
|
| Hybridization protocol |
Fragmented and labeled DNA was added to a hybridization cocktail containing, 20X Eukaryotic Hyb-Controls, 2X Hybridization mix, control oligonucleotide B2, DMSO and nuclease free water (at concentrations defined by Affymetrix Ltd). The hybridization cocktail was heated at 99°C for 5 minutes and then 45°C for a further 5 minutes in a heat block before centrifuging at 14,000g for 1 minute. Affymetrix Rat Gene 2.0 Sense Target (ST) Genechips were equilibrated to room temperature immediately before use and 130µL of hybridization cocktail was injected into the array though the septa on the reverse of the chip. The arrays were then placed in a hybridization oven (with rotating arm) at 45°C (60rpm) for 17 hours. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
Samples were scanned using the Affymetrix Genechip Scanner 3000. The scanner was turned on ~10 minutes prior to scanning to warm the laser. In the AGCC, autoloader function was selected and arrays were firstly wiped clean using a non-abrasive tissue before loading onto the scanner carousel (up to 30 at a time).
|
| Description |
Gene expression data from livers of rats treated with GSK932121A for 5 hours
|
| Data processing |
Once scanning was complete the hybridization image was inspected to ensure no gross scan-errors could be detected. CEL.files were then transferred from the PC and differential gene expression analysis was performed using Array Studio version 7.0 (www.omicsoft.com). Affymetrix data was normalised using the robust multichip average (RMA) method (Irizarry, Hobbs et al. 2003) and differentially expressed genes were defined as those with greater than 1.5 fold change in expression, at p<0.05.
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| |
|
| Submission date |
Jan 28, 2015 |
| Last update date |
Jan 29, 2015 |
| Contact name |
Ashley Broom |
| E-mail(s) |
ashleybroom88@gmail.com
|
| Organization name |
Imperial College London
|
| Street address |
Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17117 |
| Series (1) |
| GSE65374 |
Analysis of gene expression profiles from the liver of rats treated with the mitochondrial electron transport chain complex III inhibitor GSK932121A (50mg/kg) or vehicle control |
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