SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity
Standard
SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity. / Wege, Henning; Chui, Michael S; Le, Hai T; Tran, Julie M; Zern, Mark A.
in: NUCLEIC ACIDS RES, Jahrgang 31, Nr. 2, 15.01.2003, S. E3-3.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
Harvard
APA
Vancouver
Bibtex
}
RIS
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 -