Twelve six-week old wild-type (WT) C57BL/6J male mice were purchased from Charles River and 12 Pxr-/- animals (backcrossed on the C57Bl/6J background) are bred for 10y in our animal facility. Mice were acclimatized for two weeks, then randomly allocated to the different experimental groups: wild-type control (WT CONT, n=6), wild-type PCN-treated (WT PCN, n=6), Pxr-/- control (Pxr-/- CONT, n=6), Pxr-/- PCN-treated (Pxr-/- PCN, n=6). PCN-treated mice received a daily intraperitoneal injection of PCN (100 mg/kg) in corn oil for 4 days while control mice received corn oil only. Mice were killed at ZT6, 6 hours after the last PCN injection, in the fed state.
Extracted molecule
total RNA
Extraction protocol
At sacrifice, the liver, the three parts of the small intestine (duodenum, jejunum, ileum), and the colon were removed and snap-frozen in liquid nitrogen and stored at -80°C until used for RNA extraction. Total RNA was extracted with TRIzol reagent (Invitrogen).
Label
Cy3
Label protocol
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse GE v2 microarray (8X60K, Design 074809 enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
Scan protocol
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
Description
C14
Data processing
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 074809_D_F_20150624). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 14 out of 17 microarrays or with a minimal weight of 3 per group from at least one experimental group. At this step, 39394 spots out of 62976 were selected. The mean signal of the 9 first samples and 8 last ones were substracted to individuals signals to correct the batch effect of the 2 serials (characteristics:WashBatch) observed during the microarray washing procedure. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 36695 rows each corresponding to a unique ProbeName (provided as data Matrix).
