To determine telomerase activity, the telomeric repeat amplification protocol (TRAP) assay was performed as previously described.
- Kim NW
- Piatyszek MA
- Prowse KR
- Harley CB
- West MD
- Ho PLC
- Coviello GM
- Wright WE
- Weinrich SL
- Shay JW
Specific association of human telomerase activity with immortal cells and cancer.
Samples were suspended in phosphate-buffered saline (PBS), and cell pellets were recovered and resuspended in ice-cold wash buffer (10 mmol/L Hepes/KOH (pH 7.5), 1.5 mmol/L MgCl2
, 10 mmol/L KCl, 1 mmol/L dithiothreitol (DTT). After washing, the pellets were homogenized in 20 to 100 μl of ice-cold lysis buffer (10 mmol/L Tris/HCl (pH 7.5), 1 mmol/L MgCl2
, 1 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L β-mercaptoethanol, 0.5% CHAPS (Sigma Chemical Co., St. Louis, MO), and 10% glycerol). After 30 minutes of incubation on ice, the lysate was centrifuged at 15,000 × g
for 30 minutes at 4°C, and the supernatant was frozen and stored at −80°C. The protein concentration in the extract was measured by Bradford assay.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Five micrograms of protein was used for the TRAP assay. Assay tubes were prepared by sequestering 0.2 μg of CX primer (5′-CCCTTACCCTTACCCTTACCCTTAA-3′) under a wax barrier (Ampliwax, Perkin Elmer Cetus, Foster City, CA). Each extract was assayed in 50 μl of reaction mixture containing 20 mmol/L Tris/HCl (pH 8.0), 1.5 mmol/L MgCl2
, 60 mmol/L KCl, 0.005% Tween 20, 1 mmol/L EGTA, 50 μmol/L dNTPs, 0.2 μg of TS primer (5′-AATCCGTCGAGCAGAGTT-3′), 1 μg of T4g 32 protein (Boehringer Mannheim, Mannheim, Germany), and 2.5 U of Taq
DNA polymerase (Wako, Osaka, Japan). After 30 minutes of incubation at 23°C for telomerase-mediated extension of the TS primer, the reaction mixture was heated at 90°C for 3 minutes and then subjected to 31 PCR cycles of 94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 60 seconds. The PCR products were electrophoresed on a 12% polyacrylamide gel and visualized with SYBR Green I nucleic acid gel stain (FMC BioProducts, Rockland, ME). For quantitative analysis of telomerase activity, stretch PCR was used. Our method is a modified version of the original stretch PCR developed by Tatematsu et al.
- Tatematsu K
- Nakayama J
- Danbara M
- Shionoya S
- Sato H
- Omine M
- Ishikawa F
A novel quantitative 'stretch PCR assay' that detects a dramatic increase in telomerase activity during the progression of myeloid leukemias.
- Kyo S
- Takakura M
- Tanaka M
- Inoue M
Telomerase activity in cervical cancer is quantitatively distinct from that in its precursor lesions.
- Kyo S
- Takakura M
- Tanaka M
- Murakami K
- Saito R
- Hirano H
- Inoue M
Quantitative differences in telomerase activity among malignant, premalignant, and benign ovarian lesions.
Cell extracts were obtained using CHAPS buffer in the same fashion as for the TRAP assay. Five micrograms of protein extract was assayed in 50 μl of reaction mixture containing 20 mmol/L Tris/HCl (pH 8.0), 1.5 mmol/L MgCl2
, 60 mmol/L KCl, 0.005% Tween 20, 1 mmol/L EGTA, 500 μmol/L dNTPs, and 0.2 μg of TAG-U primer (5′-GTAAAACGACGGCCAGTTTGGGGTTGGGGTTGGGGTTG-3′). After 30 minutes of incubation at 30°C for telomerase-mediated extension of the TAG-U primer, telomerase products were purified by phenol/chloroform treatment performed twice, followed by ethanol precipitation. Recovered pellets were mixed with 50 μl of PCR buffer containing 20 mmol/L Tris/HCl (pH 8.0), 1.5 mmol/L MgCl2
, 60 mmol/L KCl, 0.005% Tween 20, 1 mmol/L EGTA, 50 μmol/L dNTPs, 0.2 μg of CTA-R primer (5′-CAGGAAACAGCTATGACCCCTAACCCTAACCCTAACCCT-3′), and 2.5 U ofTaq
DNA polymerase and were heated at 90°C for 5 minutes and then subjected to 25 PCR cycles of 93°C for 1 minute, 68°C for 1 minute, and 72°C for 2 minutes. The PCR products were electrophoresed on a 7% polyacrylamide gel and visualized with SYBR Green I nucleic acid gel stain. In every assay, telomerase activity was examined in various numbers of C33A cells, and a standard curve was prepared to normalize the activity. The telomerase activity in each sample was quantified by counting the staining activity of DNA ladders spanning 100 to 600 bp using NIH Image and normalized to control activity in 104
C33A cells (defined as 100 units).