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

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...

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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