TRAPeze^™ RT Telomerase Detection Kit

Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, the telomeres consist of thousands of copies of 6 base repeats (TTAGGG)(1-3). It has been suggested that telomeres protect chromosome ends since damaged chromosomes lacking telomeres undergo fusion, rearrangement and translocation (2). In somatic cells, telomere length is progressively shortened with each cell division both in vivo and in vitro (4-7) due to the inability of the DNA polymerase complex to synthesize the very 5′ end of the lagging strand (8,9).

Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3′ end of existing telomeres using its RNA component as a template (10-14). Telomerase activity has been shown to be specifically expressed in immortal cells, cancer and germ cells (15,16) where it compensates for telomere shortening during DNA replication and thus stabilizes telomere length (7,17). These observations have led to a hypothesis that telomere length may function as a "mitotic clock" to sense the number of cell divisions and eventually signal replicative senescence or programmed cell death when a critical telomere length is achieved. Therefore, expression of telomerase activity in cancer cells may be a necessary and essential step for tumor development and progression (16,18-20). The causal relationship between expression of telomerase and telomere length stabilization and the extension of the life span of the human cell has recently been reported (21).

The development of a sensitive and efficient PCR-based telomerase activity detection method, TRAP (Telomeric Repeat Amplification Protocol) (15, 22), has made possible large scale surveys of telomerase activity in human cells and tissues (15, 23-29). To date, telomerase activity has been detected in over 85% of all tumors tested spanning more than 20 different types of cancers (30-31).

The TRAPeze^™ RT Telomerase Detection Kit is a highly sensitive in vitro assay for the fluorometric detection and real time quantification of telomerase activity. It incorporates refinements to the original TRAP assay that were first introduced in the gel-based TRAPeze^™ Telomerase Detection Kit (Cat. No. S7700) and adds the ability to quantitate telomerase activity using fluorescence energy transfer (ET) primers similar to the TRAPeze^™ XL Telomerase Detection Kit (Cat. No. S7707). As in the original TRAPeze^™ Kit, primer sequence modifications that reduce amplification artifacts and PCR controls for standard curve generation are included. In addition, both the TRAPeze^™ RT and XL Kits use fluorescence energy transfer (ET) primers to generate fluorescently labeled TRAP products which permit nonisotopic, quantitative analysis of telomerase activity.

The unique design of these ET primers (Amplifluor^® primers) allows detection and quantification of telomerase activity by directly measuring real time fluorescence emission in the reaction vessels. Since Amplifluor^® primers will fluoresce only upon incorporation into the TRAP products, post-PCR sample manipulations such as electrophoretic gel or ELISA analyses are eliminated, thereby reducing the the risk of carry-over contamination. Quantitative analysis is not compromised when detection is performed in a high-throughput 96-well format unlike platforms utilizing a qualitative ELISA. Additionaly, an additional stand alone control is provided separately to assess PCR inhibitors that may be present in experimental samples. (Please see product insert for references).

The TRAPeze^™ RT Telomerase Detection Kit is a highly sensitive in vitro assay for the fluorometric detection and real time quantification of telomerase activity. It incorporates refinements to the original TRAPeze^™ assay and adds the ability to quantitate telomerase activity using fluorescence energy transfer (ET) primers.

The kit provides enough reagents to perform 224 TRAPeze^™ RT reactions.

1. CHAPS Lysis Buffer - 15°C to -25°C
2. 5X TRAPeze^™ RT Reaction Mix -15°C to -25°C
3. 5X TRAPeze^™ Control Reaction Mix 2°C to 8°C
4. PCR - Grade Water - 15°C to -25°C
5. TSR8 -15°C to -25°C
6. TSK -15°C to -25°C
7. Control Cell Pellet -75°C to -85°C

CHAPS Lysis Buffer (13.5mL)

5X TRAPeze^™ RT Reaction Mix (1.12mL)

5X TRAPeze^™ Control Reaction Mix (1.12mL)

PCR - Grade Water (8.2mL)

TSR8* (quantitation control template) (45 μL)

TSK* (pcr inhibition/normalization control) (45 μL)

Control Cell Pellet (Telomerase positive cells; 10⁶ cells)

* Caution - refer to Sec. II. Kit Components, Precautions in product insert.

Amplifluor is a registered trademark of Merck KGaA, Darmstadt, Germany

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

TRAPEZE is a trademark of Merck KGaA, Darmstadt, Germany