Non-radionuclide marker of telomerase activity in.doc

 PAGE \* MERGEFORMAT 11 Non-radionuclide marker of telomerase activity in Kim [1] using PCR technology in 1994, established a sensitive detection of telomerase - telomere repeat amplification protocol (telomerase repeat amplification protocol, TRAP). We have to be improved method of Kim set up a simple, non-radioactive silver staining method to detect brain tumors, colon cancer, esophageal cancer in the telomerase activity. One, materials and methods 1. Total protein extraction: the living tissue removed after surgery, liquid nitrogen preservation. (1) 100 ml extract containing 10 mmol / L Tris-HCl (pH 7.5); 1 mmol / L MgCl2; 1 mmol / L EGTA, 0.5 mmol / L β-mercaptoethanol; 0.5% CHAPS, the volume fraction of 10 % of glycerol. (2) organization of total protein extracted: Take about 30 mg groups using sterile ophthalmic surgical scissors as Shredded by adding 200 μl extract, 30 min after the ice bath, 4 ℃ 1 600 g centrifugation 20 min. Absorb about 150 μl supernatant under test. 2.TRAP reactions: (1) primers and diluted: RNA template reverse primer TS: 5’-AATCCGTCGAGCAGAGTT-3’18mer, MW = 5 524, the concentration of 0.541 g / L. PCR primer CX: 5’-CCTTACCCTTACCCTTACCC TAA3’24mer, MW = 7 104, a concentration of 0.688 g / L. (2) PCR amplification: ① to Kim [1] method improved to: 50 μl PCR solution containing 20 mmol / L Tris-HCl (pH8.3); 1.5 mmol / L MgCl2; 63 mmol / L KCl; 0.005% Tween-20; 1 mmol / L EGTA; 200 μmmol / L dNTP; 0.1 μgT4 g32 protein; 0.1 μg / L bovine serum albumin; 0.1 μg TS primer; 2 U Taqase; 0.5 ~ 1.0 mg total protein. ② 25 ℃ 20 min, adding 0.1 μg CX primer. ③ 94 ℃ pre-denaturation 5 min, 94 ℃ 30 s → 50 ℃ s → 72 ℃ 90 s 30 Ge through the bad, 72 ℃ extension of 10 min. 3. Electrophoresis: 12% non-denaturing polyacrylamide gel, take 10 μl PCR product, add 2 μl 6 × Lodding buffer, 150 V constant voltage 2 h. 4. Silver staining: (1) Fixed: gum in 10% glacial acetic acid, the w