SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity

TY - JOUR

T1 - SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity

AU - Wege, Henning

AU - Chui, Michael S

AU - Le, Hai T

AU - Tran, Julie M

AU - Zern, Mark A

PY - 2003/1/15

Y1 - 2003/1/15

N2 - The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.

AB - The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.

KW - Cell Line

KW - Cells, Cultured

KW - Enzyme-Linked Immunosorbent Assay

KW - Fluorescent Dyes

KW - Hepatocytes

KW - Humans

KW - Organic Chemicals

KW - Polymerase Chain Reaction

KW - Repetitive Sequences, Nucleic Acid

KW - Reproducibility of Results

KW - Sensitivity and Specificity

KW - Telomerase

KW - Telomere

KW - Tumor Cells, Cultured

M3 - SCORING: Journal article

C2 - 12527792

VL - 31

SP - E3-3

JO - NUCLEIC ACIDS RES

JF - NUCLEIC ACIDS RES

SN - 0305-1048

IS - 2

ER -