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Direct fluorescence imaging of TRAPEZE® XL reaction products from telomerase positive (lanes 1 and 2) and negative (lane 3) specimens.
TRAPeze® XL Telomerase Detection Kit
Description:
TRAPeze® XL Telomerase Detection Kit
Trade Name:
Chemicon (Millipore)
Product Overview:
The TRAPeze® XL Telomerase Detection Kit is a homogenous fluorescence assay for rapid and sensitive telomerase activity measurement in cell and tissue extracts. The TRAPeze® XL Kit incorporates the use of the novel Amplifluor™ fluorescence energy transfer-labeled primers into the original TRAPeze® Telomerase Detection Kit reaction so that quantitative measurements are obtained without radioactivity. Additionally, the unique design of Amplifluor primers enables homogeneous signal amplification and quantitation directly in the unopened PCR vessel. The closed-tube assay format eliminates the risk of contaminating unprocessed samples with PCR amplicon. The TRAPeze® XL Telomerase Detection Kit procedure does not require post-PCR manipulations such as gels or enzyme-linked immunosorbant assays (ELISAs).
Recommended Taq polymerases: must be non-proofreading, having no exonuclease activity and capable of "hot-start." Titanium Taq™, Platinum® Taq are suggested.
Recommended Taq polymerases: must be non-proofreading, having no exonuclease activity and capable of "hot-start." Titanium Taq™, Platinum® Taq are suggested.
Background Information:
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® XL Kit is a highly sensitive in vitro assay for the fluorometric detection of telomerase activity. It incorporates refinements to the original TRAP assay that were first introduced in the gel-based TRAPEZE® Telomerase Detection Kit (Cat #S7700). As in the original TRAPEZE® Kit, primer sequence modifications that reduce amplification artifacts and an internal PCR control are included. In addition, the TRAPEZE® XL Kit uses 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 fluorescence emission in the reaction vessels. Since Amplifluor® primers will fluoresce only upon incorporation into the TRAP products or the internal control, 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. In addition, inclusion of an internal control labeled with a second fluorophore serves to both monitor PCR amplification and aid in the quantitation of telomerase activity.
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® XL Kit is a highly sensitive in vitro assay for the fluorometric detection of telomerase activity. It incorporates refinements to the original TRAP assay that were first introduced in the gel-based TRAPEZE® Telomerase Detection Kit (Cat #S7700). As in the original TRAPEZE® Kit, primer sequence modifications that reduce amplification artifacts and an internal PCR control are included. In addition, the TRAPEZE® XL Kit uses 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 fluorescence emission in the reaction vessels. Since Amplifluor® primers will fluoresce only upon incorporation into the TRAP products or the internal control, 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. In addition, inclusion of an internal control labeled with a second fluorophore serves to both monitor PCR amplification and aid in the quantitation of telomerase activity.
Key Applications:
PCR
Species Reactivity:
Mammals
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Kit or Assay Type:
- Apoptosis Assays
- Cancer Kits
Components:
- CHAPS Lysis Buffer (13.5 mL)
- 5X TRAPEZE® XL Reaction Mix (1.12 mL)
- TS primer
- RP Amplifluor® primer
- K2 Amplifluor® primer
- TSK2 template
- dA, dC, dG and dTTP
- diluted in :
- 100 mM Tris-HCl, pH 8.3
- 7.5 mM MgCl2
- 315 mM KCl
- 0.25% Tween 20
- 5 mM EGTA
- 0.5 mg/mL BSA
- PCR - Grade Water (8.2 mL)
- protease, DNase, and RNase-free;
- deionized
- TSR8* (control template) (45 µL)
- 0.2 amole/µL TSR8 template
- Control Cell Pellet
- Telomerase positive cells (106 cells)
- * Caution - refer to Sec. II. Kit Components, Precautions.
Storage Conditions:
Precautions
1. Because the TRAPEZE® XL Telomerase Detection Kit detects the activity of telomerase, a RNase sensitive ribonucleoprotein, and not merely the presence of the RNA or protein components of telomerase, the assay requires enzymatically active cell or tissue samples. Furthermore, due to the sensitivity of the TRAPEZE® XL Kit assay, which can detect telomerase activity in a very small number of cells, a special laboratory setup and significant precautions are required to prevent PCR carry-over contamination and RNase contamination. These precautions are discussed in detail in Sec. V. Appendix, Laboratory Setup and Precautions and TRAPeze® XL Telomerase Detection Kit Station Setup (Area 1).
2. For Research Use Only. Not for use in diagnostic procedures.
1. Because the TRAPEZE® XL Telomerase Detection Kit detects the activity of telomerase, a RNase sensitive ribonucleoprotein, and not merely the presence of the RNA or protein components of telomerase, the assay requires enzymatically active cell or tissue samples. Furthermore, due to the sensitivity of the TRAPEZE® XL Kit assay, which can detect telomerase activity in a very small number of cells, a special laboratory setup and significant precautions are required to prevent PCR carry-over contamination and RNase contamination. These precautions are discussed in detail in Sec. V. Appendix, Laboratory Setup and Precautions and TRAPeze® XL Telomerase Detection Kit Station Setup (Area 1).
2. For Research Use Only. Not for use in diagnostic procedures.
Materials Required but Not Delivered:
Equipment and Supplies
1. Thermocycler
2. Spectrofluorometer (Option 1)
3. Fluorescence plate reader (Option 2) with appropriate filters for fluorescein and sulforhodamine detection (See Sec. V. Appendix, Excitation and Emission Filters)
4. Optically clear tubes for PCR amplification and detection, a holder for PCR tubes, or a 96-well plate if using a fluorescence plate reader (Option 2)
5. If analyzing tissues, homogenization equipment as described in Sec. III. Protocol, Extract Preparation
6. Tubes for PCR amplification and detection
7. Aerosol resistant pipette tips (RNase-free)
Reagents
1. Taq polymerase (cloned, unmodified)
2. PBS (Mg2+- and Ca2+-free)
3. Reagents for protein concentration measurement (See Sec. V. Appendix, Determination of Protein Concentration)
4. RNase inhibitor (for extract preparation from tissues)
5. Buffer used with the analytical spectrofluorometer (Option 1) (See Sec. III. Protocol, TRAPEZE® XL Telomerase Detection Kit Assay)
6. Bovuminar® Bovine Serum Albumin
1. Thermocycler
2. Spectrofluorometer (Option 1)
3. Fluorescence plate reader (Option 2) with appropriate filters for fluorescein and sulforhodamine detection (See Sec. V. Appendix, Excitation and Emission Filters)
4. Optically clear tubes for PCR amplification and detection, a holder for PCR tubes, or a 96-well plate if using a fluorescence plate reader (Option 2)
5. If analyzing tissues, homogenization equipment as described in Sec. III. Protocol, Extract Preparation
6. Tubes for PCR amplification and detection
7. Aerosol resistant pipette tips (RNase-free)
Reagents
1. Taq polymerase (cloned, unmodified)
2. PBS (Mg2+- and Ca2+-free)
3. Reagents for protein concentration measurement (See Sec. V. Appendix, Determination of Protein Concentration)
4. RNase inhibitor (for extract preparation from tissues)
5. Buffer used with the analytical spectrofluorometer (Option 1) (See Sec. III. Protocol, TRAPEZE® XL Telomerase Detection Kit Assay)
6. Bovuminar® Bovine Serum Albumin