Method for determining serum miR-224 content by using isotope dilution mass spectrometry

A mir-224, isotope dilution technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of poor accuracy of quantitative results, time-consuming and labor-intensive costs, and the results are easily affected by various factors.

Active Publication Date: 2019-09-10
AFFILIATED HOSPITAL OF NANTONG UNIV
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Problems solved by technology

The indirect method usually needs to first amplify miR-224 or modify it with chemical reagents and enzymes, and then perform gel imaging or fluorescein labeling, which takes a lot of time and labor costs, and the results are easily affected by various factors
Although the above-mentioned direct method of miR-224 solves the defects of the indirect method to a certain extent, the accuracy of quantitative results is poor

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  • Method for determining serum miR-224 content by using isotope dilution mass spectrometry
  • Method for determining serum miR-224 content by using isotope dilution mass spectrometry
  • Method for determining serum miR-224 content by using isotope dilution mass spectrometry

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Embodiment Construction

[0064] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0065] Instruments and reagents:

[0066] AB Sciex 5500 triple quadrupole mass spectrometer (USA, Applied Biosystem) was equipped with electrospray ionization source; Shimadzu Nexera X2 ultra-high performance liquid chromatography (Japan, Shimadzu Corporation) was equipped with binary high-pressure pump, automatic sample injection Instrument, column thermostat; ABN2ZA nitrogen generator (USA, PEAK company); SymmetryShield TM RP18 chromatographic column (2.1×...

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Abstract

The present invention discloses a method for determining the serum miR-224 content by using the isotope dilution mass spectrometry. The method comprises the following steps: (1) synthesizing a singlestrand of miR-224 nucleic acid to formulate into a solution, and establishing a standard curve; (2) synthesizing a nucleic acid peptide probe to formulate into the solution for capturing miR-224; (3)synthesizing the isotopically labeled polypeptide to formulate into the solution as an internal standard; (4) extracting the miRNA in a serum sample; (5) biotinylating the extracted miRNA; (6) reacting the miRNA with the streptomycin agarose sphere to form the miRNA-biotin-streptavidin agarose sphere complex; (7) adding the nucleic acid peptide probe to capture the miR-224-biotin-streptavidin agarose sphere complex; (8) after washing the complex, adding trypsin for enzymatic hydrolysis, then performing solid phase extraction on the enzymatic hydrolysate, and drying and re-dissolving the extracted polypeptide; (9)performing mass spectrometry on the dissolved polypeptide and the isotopically labeled polypeptide sample; and (10) calculating the miR-224 content in the serum sample. The methoddisclosed by the present invention has high accuracy and reliable results.

Description

technical field [0001] The invention relates to a method for measuring serum miR-224 content, in particular to a method for measuring miR-224 content by isotope dilution mass spectrometry. Background technique [0002] Studies have shown that miR-224 is a tumor-associated miRNA molecule, which is highly expressed in various malignant tumor tissues (such as gastric cancer, pancreatic cancer, liver cancer, and colorectal cancer). At present, the quantitative detection methods of miR-224 are divided into indirect method and direct method. The indirect method includes polymerase chain reaction method, fluorescence resonance energy transfer method, gene chip method, electrochemical catalytic method, next generation sequencing method, electrochemical method, etc . Direct methods include dual-specific nuclease-assisted spectroscopic detection, differential interference contrast microscopy imaging, and capillary electrophoresis. The indirect method usually needs to first amplify m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/08
CPCG01N30/02G01N30/06G01N30/08G01N2030/062
Inventor 王峰季伙燕吴安琪李袆鞠少卿王建新沈蕾
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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