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Method for detecting telomerase activity

A technology of telomerase and activity, applied in the field of detecting the activity of telomerase, can solve problems such as being unfavorable for clinical pathological detection, difficult to control reaction conditions, unable to locate tissue, etc., achieving convenient clinical detection and stem cell research, and not easy to false negative. and false positive results, strong sensitivity and specificity

Inactive Publication Date: 2003-10-22
北京科宇联合干细胞生物技术有限公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] Disadvantages: complex process, high cost, inaccurate, requires continuous observation
[0019] Disadvantages: Although PCR is relatively sensitive, the reaction conditions are not easy to control, the repeatability is poor, and false negative and false positive results are prone to occur
[0030] (1) The use of cell extracts cannot be used for tissue localization, which is not conducive to clinical and pathological detection.
[0031] Nor does it apply to research on adult stem cells;
[0032] (2) Due to the use of PCR as the basis of the experiment, false negative and false positive rates are prone to occur;
[0033] (3) For a large number of samples, the repeatability is poor and the cost is high
However, this method is limited to the detection of mRNA. At present, no one has used this method to detect hTERT mRNA expression to determine telomerase activity. At the same time, no one has selected cDNA corresponding to motif-T as a probe. Related reports on detection of telomerase activity and its splicing variation

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0058] Used in this experiment, take total RNA and utilize Trizol reagent (Promega company), M-MLV reverse transcriptase kit (GiBco company), PCR product and probe purification kit Concert TM Repid PCR Purification System (Bochinger Mannheim Company), T-easy plasmid (Promega Company), the purification kit of plasmid is High Pure PlasmidIsolation Kit For mini preparations (GiBco Company), the endonucleases used all come from Promega Company, restriction endonuclease Fragment Recovery Kit (CONCERT TM Gel ExtractionSystems) (GiBco Company), gap translation labeling kit (DIG-Nick TranslationMix), and anti-digoxigenin antibody labeled with fluorescein (Anti-Digoxigenin-Fluorescein FabFragments from sheep) are all products of Roch Company. Embodiment 1: Preparation of motif-T probe Preparation process: (1) extract total RNA from human embryonic kidney cell line 293 (human embryonic kidney cell line 293, HEK-293), use M-MLV (Moloney rat Leukemia virus (Moloney murine leukemin virus...

Embodiment 2

[0059]Experimental conclusion: It shows that the probe prepared by HEK293 cells known to have telomerase activity has telomerase activity, and the activity is relatively high. It can be used to detect whether other unknown cells have telomerase activity. Example 2: Detection of telomerase activity in HEK293 cells Experimental cells: HEK293 cells 1. Cell preparation, fixation and permeabilization (note that all solutions should be treated with RNase inhibitors) 1. Use 37°C PBS cells at room temperature , fix the cells; wherein the composition of the fixative: 4% paraformaldehyde, 5% acetic acid, 0.9% NaCl 2. wash the cells with PBS at room temperature for 30 minutes; 3. process the cells as follows before hybridization: (1) dehydration : 70%, 90% and 100% ethanol; (2) Degreasing: 100% xylene (3) Hydration: 100%, 90%, 70% (4) Rinse with PBS; 4. Increase permeability: 37°C Permeabilization solution was used to increase permeability; the composition of permeation solution: 0.1% p...

Embodiment 3

[0061] Blocking solution composition: 100Mm Tris-HCl (pH=7.5), 150mM NaCl, 0.5% (w / v) blocking agent 2. Remove the cover slip and simply wash it with the solution; 3. Add the blocking solution containing the antibody and put it in In the wet box, 45 minutes; 4. Washing; solution composition: 100Mm Tris-HCl (pH=7.5), 150mM NaCl, 0.05% Tween205. Dehydration: Dehydration: 70%, 90% and 100% ethanol; 5 minutes for each concentration 6. Air drying; 7. Embed the sample with a solution of anti-fading agent; the ratio is: glycerol: anti-fading agent=1: 9 anti-fading solution: 1M Tris-HCl (pH=7.5), 2%DABCO, DAPI (75ng / ul) 8. Observed under a fluorescent microscope (excitation wavelength is 494nm, emission wavelength is 523nm), the nucleus is blue, and the cytoplasm contains a large number of green fluorescent particles with high fluorescence intensity. Experimental conclusion: It shows that HEK293 cells have telomerase activity, and the activity is relatively high. Example 3: Detection...

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Abstract

The method for detecting activity of telomerase includes the following steps: extracting total RNA of cell, reverse trancribing total RNA into cDNA, cloning the object fragment of cDNA sequence containing motif-T, synthesizing specific probe, making in situ hybridization, then detecting that it has the transcription of hTERT mRNA or not so as to define that it has the activity of telomerase or not. Because the detection of motif-T is a structure detection, the sensitivity and specificity of its measured result are strong, so that it is an accurate and simple method for detecting activity of telomerase. Said invention has important application value for diagnosing and curing tumor and making basic research of cell system.

Description

technical field [0001] The invention relates to a method for detecting the activity of telomerase, especially a method for determining the activity of telomerase by detecting a certain structural domain in human telomerase reverse transcriptase (abbreviated as hTERT, namely human Telomerase Reverse Transcriptase). , so as to provide an accurate and simple method for the detection of telomerase activity, which is of great significance for clinical tumor diagnosis and prognosis evaluation, as well as basic research on cell lines. Background technique [0002] In the course of long-term research, people have gradually discovered telomeres - this special structure at the end of chromosomes. The important feature of this structure in biology is that it can gradually become shorter as the cell divides, and when it becomes short to a certain extent, the cell will die. Since the length of the telomere has become a signal to exit the cell cycle, people regard the length of this char...

Claims

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Application Information

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IPC IPC(8): C12Q1/25
Inventor 王亚军沈丽
Owner 北京科宇联合干细胞生物技术有限公司
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