A total of 10 male Fischer 344 rats, 5 weeks old, were purchased from Charles River Japan, Incorporated, Atsugi, Kanagawa, Japan.
More...A total of 10 male Fischer 344 rats, 5 weeks old, were purchased from Charles River Japan, Incorporated, Atsugi, Kanagawa, Japan. Rats were divided into 2 groups each consisting of 5 animals housed in a plastic cage with white flake bedding in an air-conditioned room. Rats were used for the experimentation after a 1-week acclimation on a basal diet (CRF-1, Oriental Yeast Corporation, Itabashi, Tokyo, Japan) and allowed free access to food and tap water throughout the acclimation and experimental periods. Body weight, food consumption, and water intake were monitored weekly. The CDAA diet was obtained from Dyets, Bethlehem, PA.
After acclimation, group 1 rats were administered the basal diet, while group 2 rats were placed on the CDAA diet for 70 weeks and then sacrificed. The livers were taken and macroscopically examined. Portions of group 1 livers, as well as the macroscopic tumors and their surrounding, non-cancerous tissues of group 2 livers were fixed in 10% neutrally-buffered-formalin for 24 hours, embedded in paraffin, processed for the routine hematoxylin-and-eosin staining procedure, and histologically examined. Remaining portions of these 3 types of liver tissues were immediately frozen in liquid nitrogen and stored at -80C. Group 1 liver tissues, group 2 liver tissues from non-cancerous areas, and group 2 liver tissues from tumors (after histologically diagnosed as HCCs; see the results section) were used as normal liver samples, surrounding non-cancerous liver samples, and HCC samples, hereafter referred to as CON, NC, and CA, respectively. The microarray and RT-PCR experiments and the subsequent data analysis were performed using 5 CON samples from individual group 1 rats and 5 matched pairs of NC and CA samples from individual group 2 animals.
RNA Isolation and Probe Labeling. Total RNA from liver tissues was isolated using RNeasy Midi kits (QIAGEN, Hilden, Germany). The integrity of the RNA was checked by electrophoresis on 1% agarose-formaldehyde gels. Five micrograms of total RNA from individual samples were labeled with Cy3 (Amersham Biosciences, Uppsara, Sweden) using BD Atlas Powerscript Fluorescent Labeling kits (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer’s protocols.
Hybridization, Scanning, and Quantification. Cy3-labeled probes were hybridized to Atlas Rat 3.8 I microarrays (BD Biosciences Clontech) containing 3757 genes for 16 hours at 50C. After hybridization, microarrays were washed, dried, and scanned using a GMS 418 confocal laser scanner (Genetic MicroSystems, Woburn, MA). Fluorescence intensities of the Cy3 channels were quantified using ImaGene 4.0 software (BioDiscovery, El Segundo, CA).
Data analysis was performed using the GeneSpring software, version 5.1 (Silicon Genetics, Redwood City, CA), including appropriate statistics. Dividing by the median calculated from all of the signal intensities for the Cy3 in a given sample normalized the fluorescence signal for each gene. The VALUEs for each gene was then calculated by dividing the normalized signal by the median of each gene to offset differences in expression levels between genes.
Keywords: parallel sample
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