tissue: Normal tongue tissue strain: F344 Rat gender: Male histology: Normal age: approximately 33 weeks treatment: none
Treatment protocol
NQO was purchased from Sigma-Aldrich, St. Louis, MO, and was stored in the dark at −20°C until used. NQO was administered in the drinking water at 20 ppm for a period of 10 weeks. After dose preparation, all formulations of NQO were stored in the dark at 4°C until used. Bottles containing NQO-supplemented drinking water were wrapped with foil to preclude possible photodegradation of the carcinogen, and were changed at two- to three-day intervals throughout the study. Rats received their first exposure to NQO at seven to nine weeks of age.
Growth protocol
Male F344 rats were received at six to seven weeks of age from virus-free barrier colonies that are maintained under contract to the National Cancer Institute, Frederick, MD. Rats were held in quarantine for a minimum of one week prior to study initiation; prior to randomization into a study, each rat underwent a hand-held physical examination to ensure its suitability for use as a test subject. Animals were housed on hardwood bedding in polycarbonate shoebox cages (two to three per cage) in a windowless room that was illuminated for 12 hours each day and maintained at 22 ± 1°C and within the range of 30% to 70% relative humidity. Throughout all studies, rats were permitted free access to Purina 5001 Laboratory Diet (PMI Feeds, Brentwood, MO) and City of Chicago drinking water (provided in water bottles). All food cups and water bottles were replaced a minimum of twice weekly.
Extracted molecule
total RNA
Extraction protocol
Paired sets of normal and neoplastic oral tissues were harvested from rats at 23 to 26 weeks after their first exposure to 4-NQO. At necropsy, gross lesions were collected from the tongue of 4-NQO-treated rats and bisected longitudinally. Half of each lesion was flash-frozen in liquid nitrogen for analysis of gene expression/promoter methylation. The other half of each lesion was fixed in formalin, processed using routine histologic methods, stained with H&E, and evaluated microscopically to confirm malignancy. Phenotypically normal tissue was collected from adjacent areas of the tongue of the same 4-NQO-treated rats, and was flash-frozen or fixed in a manner identical to that used for tumor tissues. Total RNA was isolated from histologically confirmed squamous cell carcinomas and adjacent phenotypically normal tissues using Qiagen’s RNeasy Mini kit by following the manufacturer’s instructions. Total RNA was used for microarray or RT-PCR analysis.
Label
Alexa-555
Label protocol
First and second strand cDNA was prepared from the total RNA samples. Labeled cRNA target was prepared from the DNA template and verified on the Bioanalyzer. cRNA was fragmented to uniform size and verified on the Bioanalyzer.
Hybridization protocol
1 µg of purified cRNA was fragmented to uniform size and applied to Agilent Whole Human Genome microarrays (Agilent Technologies, Santa Clara, CA) in hybridization buffer. Arrays were hybridized at 65° C for 17 hrs. in a rotating incubator, and washed at 37° C for 1 min.
Scan protocol
Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner (Agilent Technologies, Santa Clara, CA) at 5 µm resolution.
Data processing
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX software (Agilent Technologies, Santa Clara, CA).
Molecular characterization of 4-NQO induced F344 rat tongue carcinogenesis: alteration of multiple gene expression and hypomethylation of PTGS2 proximal promoter
Data table header descriptions
ID_REF
VALUE
Intensity values are normalized to the 75th percentile intensity of each array.
