Method for detecting telomerase activity

A technology for telomerase and activity, which is applied in the field of detection of telomerase activity, can solve the problems of probes being difficult to obtain and popularize, and achieve the effects of simple operation, improved sensitivity and specificity, and low detection limit

Inactive Publication Date: 2015-10-14
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
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Problems solved by technology

Although the transcription-mediated amplification/hybridization protection method has good specificity and high sensi

Method used

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  • Method for detecting telomerase activity
  • Method for detecting telomerase activity
  • Method for detecting telomerase activity

Examples

Experimental program
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Embodiment 1

[0060] Step 1: Preparation of Telomerase Extract

[0061] Firstly, telomerase was extracted from bladder cancer cells E-J, breast cancer cells MCF-7, cervical cancer cells HeLa and human embryonic lung fibroblasts by CHAPS method. Put the trypsin solution containing the sample cells to be tested in a centrifuge tube, centrifuge at 160g for 5min, discard the supernatant, wash the cells 3 times with PBS, centrifuge at 160g for 5min after each wash, and take the third wash Then, 10 μL of PBS suspension containing cells was counted. Add the cells to a certain volume of RNase inhibitor-treated ice-cold CHAPS lysate, the control concentration is 5000 cells / μL, put the mixture in ice for 30min, then centrifuge at 12000g for 20min at 4℃, and take the supernatant , which is the telomerase extract, which was stored at -75°C for later use. Wherein the CHAPS lysate includes 10mM Tris-HCl pH7.5, 1mM MgCl 2 , 1mM EGTA, 0.1mM phenylmethylsulfonyl fluoride (PMSF), 5mM β-mercaptoethanol, 0....

Embodiment 2

[0067] Repeat Example 1 with the same steps described above, the difference is that the samples to be tested are bladder cancer cells E-J, breast cancer cells MCF-7, and cervical cancer cells HeLa; after step 1, heat inactivation of telomerase was also carried out , that is, keeping the telomerase extract in a 94° C. water bath for 20 minutes to inactivate the telomerase; in step 2, a reaction system includes the telomerase extract equivalent to 10,000 cells.

Embodiment 3

[0069] Repeat Example 1 with the same steps described above, the difference is that step 2 is directly entered without step 1, and 2 μL of 8U / μL BstDNA polymerase, 2 μL of 0.05% trypsin, 2 μL of 1 μM thrombin, and 1 mg / mL bovine serum Albumin 2 μL, and cell lysis buffer 2 μL instead of telomerase extract.

[0070] The fluorescence detection result of embodiment 1-3 is as figure 2 shown. Among them, A is the active telomerase extracted from bladder cancer cells E-J, B is the heat-inactivated telomerase extracted from bladder cancer cells E-J, C is the active telomerase extracted from breast cancer cell MCF-7, and D is Heat-inactivated telomerase extracted from breast cancer cell MCF-7, E is active telomerase extracted from cervical cancer cell HeLa, F is heat-inactivated telomerase extracted from cervical cancer cell HeLa, G is Active telomerase extracted from human embryonic lung fibroblasts, H is cell lysis buffer, I is Bst DNA polymerase, J is trypsin, K is thrombin, L is...

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Abstract

The invention discloses a method for detecting telomerase activity. The method comprises the following steps: performing constant temperature incubation an aggregation-induced emission compound with positive groups, telomerase primers, dNTPs, an RNA enzyme inhibitor and a to-be-detected sample in a telomerase amplification buffer at the temperature of 23-37 DEG C so that the telomerase primers in a reaction system generate a telomerase extension reaction, and inactivating the reaction system at the temperature of 80-100 DEG C to stop the extension reaction. As a DNA molecule phosphoric acid skeleton carries negative charges, with the extension of telomerase primer sequences, the amount of aggregation-induced emission compounds capable of being bonded on each chain can be increased, the fluorescence effect of a reaction solution can be enhanced, and the telomerase activity of the to-be-detected sample can be detected by detecting the fluorescence intensity of the reaction solution. According to the invention, specific detection on the telomerase activity can be realized.

Description

technical field [0001] The invention belongs to the field of biological detection, and more specifically relates to a method for detecting telomerase activity. Background technique [0002] The ends of chromosomes in eukaryotes have telomeres, which are complexes of repetitive DNA sequences and proteins. Telomeres play an important role in cell chromosomes, which can maintain the integrity and stability of chromosomes, and their length reflects the potential of cells to continue to replicate. Telomeres prevent the natural loss of DNA base pairs at the ends of chromosomes from affecting the structure of meaningful genetic sequences. Therefore, as the cell division continues, the telomeres of normal cells will continue to shorten, and eventually too short telomeres will make the cells unable to continue to divide, thereby limiting the lifespan of the cells and preventing excessive cell division. [0003] In 1984, Ssampay et al. discovered a ribonucleoproteinase composed of a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/6853C12Q2563/107C12Q2565/101C12Q2521/113
Inventor 夏帆庄原娄筱叮
Owner HUAZHONG UNIV OF SCI & TECH
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