Human Telomerase Activity Detection Kit (Probe qPCR Method)

Description

Telomerase is present in 85-90% of tumor tissues. Therefore, it is possible to diagnose most tumors at an early stage by detecting telomerase activity. Currently, the most commonly used method for detecting telomerase activity is TRAP (Telomeric Repeat Amplification Protocol). It takes advantage of the characteristic that telomerase can add different numbers of TTAGGG sequences to the end of the substrate DNA, and efficiently detects telomerase activity by detecting the extension products through PCR. However, the traditional TRAP method is an end-point electrophoresis detection method, which has low sensitivity, cannot be used for quantification, and is also prone to aerosol contamination.

Synonym(s):

TRAP Assay

Features

• Ready-to-use: All reagents from cell lysis to qPCR are provided, eliminating the need to optimize the conditions by yourself.

• Based on probe-based fluorescence quantitative PCR, it has higher sensitivity than the electrophoresis-based TRAP method.

• Using probes, it has higher specificity than the electrophoresis-based TRAP method.

• The primers and probes have been optimized, with high sensitivity, and the minimum detection limit can reach 100 copies per reaction.

• A positive control is provided, allowing for quantitative analysis based on this control.

• One-tube operation, eliminating the worry of aerosol contamination.

• It can be used for both quantification and qualitative analysis. When used for quantification, the linear range is at least 5 orders of magnitude.

• It can be used not only for cultured cells but also for solid tissues (including tumor tissues).

• This product is sufficient for 50 TRAP PCR detections in a 20 uL reaction system.

• This product is only intended for scientific research purposes.

Specification and ingredient list

 

Note: Add 55 uL ultra-pure water to the lyophilized substrate and 165 uL to the primers-probe mix before first use. Vortex for 1 minute each, centrifuge briefly, and place on ice. Store unused aliquots at -20deg C.

Reagents to Prepare Separately

• BCA Protein Assay Kit

• Solid-Phase RNase Scavenger

Storage

• Ship at low temperature; store at -20deg C.

• Valid for 24 months from manufacture date.

Protocol

1. Standard Curve Preparation

Prepare 10-fold serial dilutions (1E1 to 1E6 copies/uL) from the 1E7 copies/uL positive control in a dedicated area to avoid contamination:

1. Label 6 tubes (1 to 6).

2. Add 45 uL dilution buffer to each tube.

3. Add 5 uL positive control to Tube 6, vortex 1 minute ‚Üí 1E6 copies/uL.

4. Transfer 5 uL from Tube 6 to Tube 5, vortex ‚Üí 1E5 copies/uL. Repeat for Tubes 4-1 to obtain 1E4 to 1E1 copies/uL.

5. Keep on ice until use.

2. Telomerase Extraction

Caution: Telomerase contains RNA vulnerable to degradation. Use RNase scavenger to clean work surfaces and operate at low temperatures.

For Frozen Solid Tissues:

Grind 50-100 mg frozen tissue in liquid nitrogen, transfer to a pre-chilled glass homogenizer. Add 200 uL ice-cold lysis buffer, homogenize 6 times gently, ice-bath for 30 minutes (vortex every 5 minutes). Proceed to Step 10.

Note: Check cell lysis under a microscope. Reduce lysis buffer proportionally for smaller tissue samples. Tissues stored at -20deg C lose activity after 2 months; -80deg C storage preserves activity for years.

For Fresh Cultured Cells/Tissues:

Wash 1E6 trypsinized cells or 50-100 mg trypsinized tissue with ice-cold PBS, centrifuge at 3000g 4deg C for 5 min. Resuspend pellets in 200 uL lysis buffer:

Cells: Vortex 10 sec, ice-bath 30 min (vortex every 5 min).

Tissues: Homogenize on ice, ice-bath 30 min (vortex every 5 min).
Scale lysis buffer for smaller samples.

Centrifuge at 14000g 4deg C for 20 min. Collect 160 uL supernatant (telomerase-containing); discard the remaining 40 uL.

Measure total protein concentration using the BCA kit.

Adjust protein concentration to 3 ug/uL with lysis buffer. Aliquot: use immediately on ice, store excess at -80deg C (stable for 1 year). These are your telomerase test samples.

Prepare heat-inactivated negative controls: heat 1 aliquot of test sample at 95deg C for 10 min, chill on ice. Store unused controls at -80deg C (stable for 1 year).

3. Probe-based TRAP (20 uL Reaction)

Ensure consistent protein amount/cell number per reaction for valid comparisons.

Set up reactions (for N samples, prepare 2N+6 tubes):

 

Mix by pipetting, transfer to qPCR machine. Run the following program:

 

4. Data Analysis

Validity Check:

If standard curve samples show no Ct or Ct ‚â•35 (FAM signal negative), or negative control shows Ct <35 (FAM signal positive), the experiment is invalid. Repeat or contact technical support.

Standard Curve:

• Plot log(standard concentration) vs. Ct (FAM channel). The correlation coefficient (r¬≤) must exceed 0.95.

• Calculate target telomere repeat copies from sample Ct values using the standard curve. Telomerase activity correlates with the number of new telomere repeats synthesized.

Related Products

Telomere Length Detection Kit