For control/normal diet group, F344 rats were fed with basal rodent chow (CE-2; CLEA Japan Inc., Tokyo, Japan) and control dehydrogenized water (CDW) in air for 3 weeks. For hydrogen-treated/normal diet group, F344 rats were fed with basal rodent chow and hydrogen-rich water (HRW) in hydrogen-containing air (HCA; 2% hydrogen/98% air) for 3 weeks. For control/CDAA diet group, F344 rats were fed with a choline-deficient L-amino acid-defined diet (CDAA diet; A06083101, Research Diets, Inc., New Brunswick, NJ) and CDW in air for 3 weeks. For hydrogen-treated/CDAA diet group, F344 rats were fed with CDAA diet and HRW in HCA for 3 weeks. HRW (0.7mM dissolved hydrogen) was generated from distilled water with 0.44 mM Na2SO4 using Aquela Blue, a water-electrolyzing device to produce electrolyzed hydrogen-saturated water near neutral pH (MiZ Co., Ltd, Fujisawa, Japan). The CDW was prepared by gently stirring hydrogen-rich water in open air for 24 hours.
Extracted molecule
total RNA
Extraction protocol
Livers from rats of four groups were immersed in RNAlater solution (Ambion, Austin, TX) for several days at 4°C and then total RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) from the rats.
Label
biotin
Label protocol
Comparative analysis of gene expression profiles was performed using the RG-230 PM Array Strips and the Affymetrix GeneAtlas microarray system (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's instructions. For RNA samples from the rat livers, the corresponding amplified and biotin-labeled antisense RNA (aRNA) was synthesized using a GeneChip 3'IVT Express kit (Affymetrix). The resulting aRNA was fragmented as described by the manufacturer.
Hybridization protocol
The biotin-labeled aRNAs were hybridized with the array strips at 45°C for 16 h. After hybridization, the strips were washed and stained in the GeneAtlas Fluidics Station using the GeneAtlas Hybridization, Wash, and Stain Kit (Affymetrix).
Scan protocol
The intensity of each hybridized probe was measured using the GeneAtlas Imaging Station.
Description
SAMPLE 5
Data processing
The obtained CEL files were normalized and summarized using the robust multiarray average (RMA) method with Expression Console software (Affymetrix).
