Total RNA of tissue samples and cell lines were isolated by using Trizol reagent according to the instruction manual (Invitrogen). Total RNA of leukocytes obtained from 2 ml selleck inhibitor of peripheral blood was isolated by using PURESCRIPT RNA Isolation Kit (Gentra systems). RT-PCR Five microgram of the total RNA was reverse transcribed using oligo-dT primer and SuperScript III (Invitrogen) according to the instruction manual. To confirm the expression of Rad18, primer sets, 5′-TTC, ACA, AAA, GGA, AGC, CGC, TG

(VX-680 cost forward) and 5′-TTA, CTG, AGG, TCA, TAT, TAT, CTT, C (reverse) were used to amplify 310 bp region of human Rad18 gene. PCR was carried out in a condition of, 3 min at 94°C for initial denaturing, followed by 35 cycles of amplification (94°C for

30 sec, 55°C for 30 sec, and 72°C for 30 sec) using GoTaq (Promega). The amplified products were visualized PRI-724 chemical structure on 1.2% agarose gel with ethidium bromide. GAPDH in the same samples was also amplified using 25 cycles PCR reaction as the internal control. The primer sets for GAPDH is 5′-TGA, CCA, CAG, TCC, ATG, CCA, TC (forward) and 5′-CCA, CCC, TGT, TGC, TGT, AGC, C (reverse). Fragment Southern Genomic DNA from human breast cancer cell line MCF7 and lung carcinoma cell line PC3 were isolated using TRIZOL according to the instruction manual. MCF7 was used as positive control which was confirmed that this cell line carry wild type Rad18 by RT-PCR direct sequencing (data not shown). Ten microgram of genomic DNA were digested by EcoRI or HindIII, electrophoresed on a 0.8% agarose gel and transferred to a Hybond-NX membrane (Amersham).

Full length cDNA clone of Rad18 was labeled using Psoralen-Biotin nonisotopic labeling kit (BrightStar) and hybridized in PEG-SDS including 100 μg/ml Salmon sperm DNA at 65°C. Detection was done using BioDetect nonisotopic detection kit (BrightStar) according to the instruction manual. Membrane was exposed to X-ray film and developed. RT-PCR SSCP and direct sequencing PJ34 HCl The primer sets for RT-PCR SSCP are shown in Table 1. Each primer sets were designed to partially overlap the next fragment with the length not more than 200 bp. Ten primer sets cover the whole open reading frame of Rad18 gene and partially, 5′ and 3′ non coding lesion. PCR condition is, 3 min at 94°C for initial denaturing, followed by 35 cycles of amplification (94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec). Each sample was denatured 5 min at 95°C and rapidly chilled on ice and loaded into 10% acrylamide gel including 5.4% glycerol for 6 hours at 120V using MiniProtean3 (BioRad) at 4°C. After electrophoreses, gels were stained using Silver Stain Plus Kit according to the instruction manual (BioRad). All samples were screened for the presence of an aberrant band compared with reference sample. Samples with abnormal SSCP bands were directly sequenced by ABI 310. Cycle sequencing was performed using Big-Dye Terminator v3.1 (Applied Biosystems).