5% carboxymethyl cellulose (20 mg/1 ml vehicle). Induction
of liver carcinogenesis Induction of liver carcinogenesis was carried out according the following protocol: each rat received Selleckchem 4-Hydroxytamoxifen an oral dose of 20 mg/kg (NDEA/weight), for 9 weeks (5 days/week) followed by another oral dose of 10 mg/kg (NDEA/weight) for 6 weeks (5 days/week). Experimental groups Rats were acclimatized for 4 days before carrying out the experimental work. Animals were divided into 3 groups: the 1st group (14 animals) was treated with NDEA for 15 weeks as detailed above and designated as (NDEA-treated), the 2nd group (12 animals) was treated simultaneously with NDEA (20 mg/kg for 9 weeks followed by 10 mg/kg for 6 weeks) and Quercetin in a dose of 200 mg/kg daily, for 15 weeks as detailed above, the 3rd group of rats (10 animals) was used as control (oral dose of saline was administered). At the end of the experimental period, rats were food-deprived overnight and were killed by cervical decapitation. The liver was immediately excised, rinsed with ice-cold saline and blotted dry and accurately weighed. A small portion of liver was fixed in 10% formal-saline for the histopathological studies. DNA extraction and amplification of RAPD markers Genomic DNA was extracted from
liver samples using Wizard Genomic DNA Purification kit (Promega, Madison, USA) following the manufacturer’s EPZ5676 nmr instructions. DNA was visualized on a 0.7% agarose gel. Quality and concentration of DNA were determined
spectrophotometrically. Three random primers were used to study the genetic difference between the examined animals. The primers used in this study are listed in Table 1. Optimization of PCR conditions for ultimate discriminatory power was achieved. RAPD-PCR was carried out in a 25 μl total reaction volume containing 2.5 μl 10× buffer, 0.2 mM dNT’Ps, 100 pmol primer, 2 U Taq DNA polymerase, 3.0 mM MgCl2, 50 ng DNA template and nuclease-free water. The amplification program used was 4 min at 94°C (hot start), 1 min at 94°C, 1 min at 30°C and 1 min at 72°C for 36 cycles followed by one cycle of 72°C for 10 min. PCR amplification was carried out in a DNA Alpelisib molecular weight thermal cycler (Model 380 A, Applied Biosystems, CA, USA). PCR products were Glutathione peroxidase visualized on 2% agarose gel. Table 1 Arbitrary primer sequences used in this study Primer name Primer sequence EZ 5′-GCATCACAGACCTGTTATTGCCTC-3′ Chi 15 5′-GGYGGYTGGAATGARGG-3′ P 53 F 5′-CATCGAATTCTGGAAACTTTCCACTTGAT-3′ P 53 R 5′GTAGGAATTCGTCCCAAGCAATGGATGAT-3′ Specific PCR assay for polymorphism of p 53 gene For the p53 PCR, DNA of control, hepatic carcinoma and quercetin-treated samples was used up for the p53 -specific PCR assays. A primer set (Forward: 5′-CAT CGA ATT CTG GAA ACT TTC CAC TTG AT-3′ and Reverse: 5′-GTA GGA ATT CGT CCC AAG CAA TGG ATG AT-3′) was used for detection of p53 sequence.