Total RNA from small intestine of wild type or Cyp3a-/- mice was prepared using ISOGEN (Nippon Gene, Tokyo, Japan) and treated with RNase-free DNase I (Wako Pure Chemicals, Osaka, Japan), and were then purified using RNeasy columns (Qiagen, Hilden, Germany), in accordance with the manufacturer’s instructions.The concentration of the obtained total RNA was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA quality was determined using the RNA ScreenTape and Reagent Kit (Agilent Technologies, Palo Alto, CA, USA) and an Agilent 2200 TapeStation (Agilent Technologies), and RNA was stored in RNase-free water at -80ºC.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 μg total RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA, USA). Dye incorporation and cRNA yield were checked with the Nanodrop spectrophotometer.
Hybridization protocol
Cy3-labeled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 μl containig 10×Blocking Agent, Nuclease-free water, and 25×Fragmentation Buffer following the manufature's instructions. On completion of the fragmentation reaction, 55 μl of 2×GE Hybridization Buffer (HI-RPM) was added to the fragmentation mixture and hybridized to 44K Agilent Whole Human Genome Oligo Microarays (G4846A) containing 44,397 features representing 39,429 biological probes for 17 hours at 65ºC in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minutes at room temparature with GE Wash Buffer 1 and 1 minute with 37°C GE Wash Buffer 2 (Agilent Technologies).
Scan protocol
After washing microarrays according to manufacture's instructions, microarray slides were scanned immediately on the Agilent DNA Microarray Scanner (G4900DA) using one color scan setting for 4×44K array slides (Scan Area 61×21.6 mm, Scan resolution 5 μm, Dye channel is set to green, and 20 bit scan mode (no XDR scan mode)).
Data processing
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent Technologies) using default parameters (protocol GE1_1105_Oct12 and Grid: 026655_D_F_20140728). The data from all microarrays were analyzed using GeneSpringGX 14.5 (Agilent Technologies). After data transformation (to convert any negative value to 0.01), Intensity value of green Processedsignal from the Feature Extraction was normalized per chip to the 75th percentile.
