tissue: liver genotype: wild type age: 9 to 11 weeks treatment: Phenobarbital time point: 7 days donorid: 2006
Treatment protocol
Phenobarbital (PB; free acid, >99.0%, Sigma, St Louis, MO, no. 04710, 0.05% (wt/vol) in drinking water) was administered to one group through ad libitum access to drinking water, as previously reported (see methods in PMID: 24690595). Mice were reated with PB starting at 8 weeks of age. PB was adminis- tered via the drinking water (PB solution prepared freshly every fourth day) at concentrations of 0.005, 0.01, 0.02, and 0.05% (wt/vol). Mice were kept on a 12 h dark/light cycle and had ac- cessed to food and water ad libitum. All animals were sacrificed between 9 and 11 a.m. to avoid circadian influences.checked daily for activity and behavior and sacrificed on the indicated dates. Liver (incl. left, caudal and median parts) were sampled. To ensure sample homo- geneity for different molecular profiling methods, frozen liver samples were reduced to powder with CovarisCryoprep (Co- varis Inc., Woburn, MA) system and aliquoted on dry ice. For the 4-week dose response study, mice (n = 4 per group) were treated with PB starting at 8 weeks of age. PB was adminis- tered via the drinking water (PB solution prepared freshly every fourth day) at concentrations of 0.005, 0.01, 0.02, and 0.05% (wt/vol). Mice were kept on a 12 h dark/light cycle and had ac- cessed to food and water ad libitum. All animals were sacrificed between 9 and 11 a.m. to avoid circadian influences.
Growth protocol
C57BL/6 male wild-type, knock-out CARKO -PXRKO and humanized CARh - PXRh mice were obtained from TaconicArtemis (Germany). For the 13-week time course study, 9–11 week-old mice (age selected to avoid the confounding effect of liver maturation observed in younger animals) were allowed to acclimatize for 5 days prior to being randomly divided into two treatment groups (n = 5 per time point).
Extracted molecule
total RNA
Extraction protocol
Frozen liver samples were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) and subsequently purified on a silica-gel-based-membrane (RNeasy, Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. RNA quality was assessed by measuring the RIN (RNA Integrity Number) using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA was stored at −80◦C. miRNA was quantified using the Rediplate Ribogreen RNA quantitation kit (Life Technologies).
Label
biotin
Label protocol
Processing of miRNA 2.0 experiments was conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Santa Clara, CA).
Hybridization protocol
Processing of miRNA 2.0 experiments was conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Santa Clara, CA).
Scan protocol
Processing of mi RNA 2.0 experiments was conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Santa Clara, CA).
Affymetrix mi-croRNA chips were preprocessed and normalized according to the Affymetrix miRNA QCTool manual. Briefly, the back- ground control probes of the chips were grouped into bins of same dinucleotide Guanine-Cytosine (GC) content. The median signal of the background bin that matches with the GC con- tent of the probe was then subtracted from the probe signal. The background corrected probes (for all probes on the chip in- cluding those of other species) were quantile normalized across chips, log2 transformed and summarized into probe sets with the median polish method as in standard RMA (Bolstad et al., 2003). We floored all normalized signal values to 1.0.
