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Kit for detecting activity degree of telomerase in whole blood or serum sample

A telomerase and kit technology, applied in the field of molecular biology, can solve the problems of patient inconvenience, low detection sensitivity, missed diagnosis of patients, etc., and achieve the effects of saving samples, short analysis time and high sensitivity

Inactive Publication Date: 2015-05-06
SHANGHAI DIAN CLINICAL TESTING CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, this detection method in the prior art is not perfect, and has the disadvantage of low detection sensitivity, which often leads to missed diagnosis of some patients, or "false positive" occurs, which brings inconvenience to patients

Method used

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  • Kit for detecting activity degree of telomerase in whole blood or serum sample
  • Kit for detecting activity degree of telomerase in whole blood or serum sample
  • Kit for detecting activity degree of telomerase in whole blood or serum sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation method of a kit for detecting the activity of telomerase in whole blood or serum samples.

[0051] 1. Total RNA extraction reagents:

[0052] (1) Cell lysate Trizol: measure 500mL redistilled phenol, 250g guanidine isothiocyanate, 17.6mL 0.75mol / L sodium citrate solution (pH≥7), 26.4mL 100g / L lauryl sarcosine Sodium solution, 50mL 2mol / L sodium acetate solution (pH ≥ 4), 293mL distilled water, mix well and store at 4°C;

[0053] (2) extractant: chloroform;

[0054] (3) Precipitating agent: isopropanol;

[0055] (4) Rinse solution: Measure 75mL of absolute ethanol and dilute to 100mL;

[0056] (5) RNA solvent: distilled water to remove RNase, store at -20°C.

[0057] 2. Reagents for reverse transcription:

[0058] (1) Reverse transcription reaction buffer: weigh 302.95mg Tris, 279.60mg KCl, 30.50mg MgCl 2 , 77.13mg DTT, add 9mL ddH 2O, adjust the pH to 8.3 with 1mol / L HCl, dilute to 10mL, aliquot into 1mL / tube, and store at -20°C;

[0059] ...

Embodiment 2

[0075] Example 2: Extraction of whole blood / serum total RNA.

[0076] (1) Homogenization: draw 0.25mL whole blood / serum and add 0.75mL Trizol, pipette the liquid sample several times with a sample gun to help lyse the cells in the sample.

[0077] (2) Isolation stage: the homogenate sample was incubated at 15-30° C. for 5 minutes to completely decompose the ribosomes. Add 0.2mL extract for every 0.75mL Trizol. Cap the sample tube tightly, shake the tube vigorously by hand for 15 s and incubate at 30°C for 15 min. Refrigerate and centrifuge at a high speed of no more than 12,000xg for 15 minutes at 2-8°C. The mixture was separated into three layers after centrifugation: a lower phenol-chloroform layer, an intermediate layer, and an upper colorless aqueous layer. RNA exists in the water sample layer without exception. The capacity of the aqueous layer was approximately 70% of the capacity of the added Trizol.

[0078] (3) RNA precipitation: transfer the water sample layer t...

Embodiment 3

[0081] Example 3: Reverse transcription reaction.

[0082] Please prepare the reaction solution on ice. In order to ensure the accuracy of the preparation of the reaction solution, when performing various reactions, the Master Mix should be prepared according to the number of reactions + 2, and then 10 μL should be dispensed into each reaction tube. The various components and practical amounts in the reaction solution are shown in Table 1, and the reverse transcription reaction was performed immediately after gentle mixing.

[0083] Table 1 Reagents and dosage in the reaction solution of reverse transcription

[0084]

[0085] Incubate at 42°C for 60min, at 70°C for 15min, and place at 4°C.

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Abstract

The invention provides a kit for detecting activity degree of telomerase in a whole blood or serum sample, belonging to the field of molecular biology detection. The kit comprises the following primers: (1) primer sequences of telomerase hTERT genes: hTERT-F: 5'-GGCCGATTGTGAACATGGA-3'; hTERT-R: 5'-CCTCTTTTCTCTGCGGAACGT-3'; and (2) probe sequences of telomerase hTERT genes: hTERT-P: 5'-TACGTCGTGGGAGCCA-3', the 5' end is connected with fluorescence reporter genes, and the 3' end is connected with fluorescence quenching genes. The kit for detecting activity degree of telomerase in the whole blood or serum sample has the advantages of high detection sensitivity, short analysis time and high stability.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a kit for detecting the activity degree of telomerase in whole blood or serum samples. Background technique [0002] Telomerase is a ribonucleoprotein complex composed of RNA and protein, which has reverse transcriptase activity. Its components include three parts: telomerase RNA template (human telomerase RNA, hTR), telomerase associated protein 1 (human telomerase associated protein 1, hTEP1) and telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT). Studies have shown that hTERT is the core of telomerase, only expressed in telomerase-positive cells, and is the rate-limiting factor of telomerase, and its mRNA expression level directly determines the activity of telomerase. [0003] Normal human somatic cells generally have no telomerase activity, but 85% to 90% of malignant tumors can detect telomerase activity. In human cells, telomerase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2563/107C12Q2545/113
Inventor 赵冬梅于嘉屏
Owner SHANGHAI DIAN CLINICAL TESTING CENT
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