A method for measuring serum miR-224 content by isotope dilution mass spectrometry

Abstract

The invention discloses a method for measuring serum miR-224 content by isotope dilution mass spectrometry, which comprises the following steps: (1) synthesizing a miR-224 nucleic acid single strand and making it into a solution, and establishing a standard curve; (2) synthesizing a nucleic acid peptide probe And make a solution to capture miR-224; (3) synthesize isotope-labeled polypeptide and make a solution as an internal standard; (4) extract miRNA in serum samples; (5) biotinylate the extracted miRNA; (6) react the above miRNA with streptomycin-agarose balls to form miRNA-biotin-streptavidin-agarose ball complexes; (7) add nucleic acid peptide probes to capture miR-224-biotin-streptavidin Avidin-Sepharose sphere complex; (8) Clean the above complex and add trypsin for enzymatic hydrolysis, then solid-phase extract the enzymatic hydrolyzate, dry the extracted polypeptide and redissolve; (9) reconstitute The dissolved polypeptide and isotope-labeled polypeptide samples were subjected to mass spectrometry analysis; (10) the miR-224 content in serum samples was calculated. The 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...

AI Technical Summary

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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