Sprague Dawley CD, Approximately 8 wks at the onset of dosing, Vehicle Treated, Quad
Extracted molecule
total RNA
Extraction protocol
tissues were collected for RNA extraction (snap frozen in liquid nitrogen). RNA was extracted from tissues using a combination of TRIzol RNA extraction (Invitrogen Life Technologies, Carlsbad, CA) with the RNeasy RNA extraction kit (Qiagen, Valencia, CA). Briefly, tissue was incubated in TRIzol reagent (1 ml/100 mg tissue) for 15 s at room temperature and homogenized with a Polytron homogenizer followed by an ~ 5-min room temperature incubation. After the addition of 100 µl chloroform, 500 µl of homogenate was mixed by shaking for 15 s, incubated at room temperature for 2–3 min, and centrifuged at 10,000 x g for 10 min at 2–8°C. The supernatant was used as the input material for the RNeasy RNA extraction kit and RNA isolated according to the manufacturer's protocol. Following isolation, RNA quantity, purity, and quality were determined using a SpectraMax Plus384 (Molecular Devices, Sunnyvale, CA) spectrophotometer and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
Cy3- or Cy5-labeled cRNA was created from total RNA using reverse transcription followed by in vitro transcription and a two-step label incorporation method (Hughes et al., 2001, PMID: 11283592).
Sprague Dawley CD, Approximately 8 wks at the onset of dosing, Fenofibrate; Wy-14,643; bezafibrate; rosiglitazone; all trans retinoic acid, Quad
Extracted molecule
total RNA
Extraction protocol
same as for channel_1
Label
Cy5
Label protocol
same as for channel_1
Hybridization protocol
All treated individual samples were hybridized against a pool of RNA from time-matched (concurrent) control animals (e.g., animals treated for 3 days were hybridized against a pool of animals dosed for 3 days with vehicle). The ratio of individual animal expression to control pool expression was used for all data analysis. All hybridizations (Hughes et al., 2001, PMID: 11283592) were performed in duplicate, with fluor reversal (Cy3 or Cy5) in the second hybridization.
Scan protocol
Arrays were scanned using a DNA microarray scanner (Model G2565AA; Agilent Technologies), and feature intensities (background subtracted) were determined using feature extraction software developed at Rosetta.
Description
Quad_d06_ vehicle control pool_000.0mkd_n=11 vs. Quad_d06_Fenofibrate_400.0mkd_#1157
