CN110220994B - Method for determining content of miR-224 in serum by isotope dilution mass spectrometry - Google Patents

Method for determining content of miR-224 in serum by isotope dilution mass spectrometry Download PDF

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CN110220994B
CN110220994B CN201910599052.8A CN201910599052A CN110220994B CN 110220994 B CN110220994 B CN 110220994B CN 201910599052 A CN201910599052 A CN 201910599052A CN 110220994 B CN110220994 B CN 110220994B
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CN110220994A (en
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王峰
季伙燕
吴安琪
李袆
鞠少卿
王建新
沈蕾
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Affiliated Hospital of Nantong University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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Abstract

The invention discloses a method for determining content of serum miR-224 by using isotope dilution mass spectrometry, which comprises the following steps: (1) synthesizing miR-224 nucleic acid single chains, preparing a solution, and establishing a standard curve; (2) synthesizing a nucleic acid peptide probe and preparing a solution for capturing miR-224; (3) synthesizing isotope-labeled polypeptide and preparing solution as an internal standard; (4) extracting miRNA from a serum sample; (5) biotinylating the extracted miRNA; (6) reacting the miRNA with streptomycin agarose spheres to form a miRNA-biotin-streptavidin agarose sphere compound; (7) adding a nucleic acid peptide probe to capture a miR-224-biotin-streptavidin agarose ball compound; (8) cleaning the compound, adding trypsin for enzymolysis, then carrying out solid-phase extraction on an enzymolysis product, drying the extracted polypeptide by blowing and redissolving; (9) carrying out mass spectrometry on the redissolved polypeptide and the isotope-labeled polypeptide sample; (10) and calculating the content of miR-224 in the serum sample. The invention has high accuracy and reliable result.

Description

Method for determining content of miR-224 in serum by isotope dilution mass spectrometry
Technical Field
The invention relates to a method for measuring miR-224 content in serum, in particular to a method for measuring miR-224 content by isotope dilution mass spectrometry.
Background
Research shows that miR-224 is a miRNA molecule related to tumors and shows high expression in various malignant tumor tissues (such as gastric cancer, pancreatic cancer, liver cancer and colorectal cancer). At present, the quantitative detection method of miR-224 comprises an indirect method and a direct method, wherein the indirect method comprises a polymerase chain reaction method, a fluorescence resonance energy transfer method, a gene chip method, an electrochemical catalysis method, a next generation sequencing method, an electrochemical method and the like. The direct method comprises a dual specificity nuclease auxiliary spectrum detection method, a differential interference contrast microscope imaging method, a capillary electrophoresis method and the like. Indirect methods typically require amplification of miR-224 or modification with chemicals and enzymes followed by gel imaging or fluorescein labeling, are time and labor intensive, and results are susceptible to a variety of factors. Although the direct miR-224 method overcomes the defects of the indirect method to a certain extent, the accuracy of the quantitative result is poor.
Isotope dilution mass spectrometry is a novel miR-224 direct quantitative detection method, and is a currently accepted authoritative method for accurately quantifying compounds. The method utilizes mass spectrometry to determine the peak area or peak height ratio of a mark to a non-mark object to be detected in a sample to be detected added with an isotope mark to calculate the concentration of the object to be detected in the sample. The method has the advantages of high specificity, high sensitivity, accurate and reliable quantitative result, wider dynamic measurement range, higher traceability of the quantitative result and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a safe and reliable miR-224 content determination method, which can be used for directly determining the miR-224 content in a serum sample.
A method for determining miR-224 content by isotope dilution mass spectrometry comprises the following steps:
(1) preparing the synthesized miR-224 nucleic acid single strands into a solution for establishing a standard curve;
(2) synthesizing a nucleic acid peptide probe to prepare a solution for capturing miR-224;
(3) marking the synthesized isotope with polypeptide and preparing solution as internal standard;
(4) extracting miRNA from a serum sample;
(5) biotinylating the extracted miRNA;
(6) reacting the biotinylated miRNA with streptomycin agarose spheres to form a miRNA-biotin-streptavidin agarose sphere compound;
(7) adding a nucleic acid peptide probe to capture a miR-224-biotin-streptavidin agarose ball compound;
(8) cleaning a nucleic acid peptide probe-miR-224-biotin-streptavidin agarose bead compound, adding trypsin for enzymolysis, performing solid phase extraction on an enzymolysis product, drying and redissolving extracted polypeptide;
(9) carrying out mass spectrometry on the redissolved polypeptide and the isotope-labeled polypeptide sample;
(10) and calculating the content of miR-224 in the serum sample.
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: the synthetic amount of the miR-224 nucleic acid single strand is 4OD value, the 1OD value of each tube is subpackaged, 250 mu L DEPC water is accurately added into each tube to prepare miR-224 solution with the concentration of 20 mu mol/L and subpackaged into 1.5mL centrifuge tubes, 50 mu L of each tube is subpackaged, then 950 mu L of ultrapure water is added into each tube to be diluted to the concentration of 1 mu mol/L to be used as stock solution and subpackaged into 1.5mL centrifuge tubes, 20 mu L of each tube is subpackaged, and the subpackaged nucleic acid peptide probe solution is placed in a refrigerator at-70 ℃ for storage; before use, DEPC water was added to the miR-224 stock solution to dilute the same into a series of concentrations for establishing a standard curve.
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: the nucleic acid peptide probe sequence is 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG2-GDRALFVGEPNR, wherein 5'-CTAAACGGAACCACTAGTGACTTGA-3' (SEQ ID NO:1) is a probe nucleic acid part which is complementarily paired with miR-224 sequence 5'-UCAAGUCACUAGUGGUUCCGUUUAG-3' (SEQ ID NO: 2); GDRALFVGEPNR is a probe peptide portion characterized by a non-human any protein pancreatin hydrolyzed specific peptide segment comprising a pancreatin hydrolysis site arginine R; PEG2 (polyethylene glycol) links nucleic acids to peptides; the synthesis amount of the nucleic acid peptide probe is 20nmol, 1mL of ultrapure water is accurately added to prepare a nucleic acid peptide probe solution with the concentration of 20 mu mol/L, each tube is subpackaged with 100 mu L, then 900 mu L of ultrapure water is added into each tube to be diluted to the concentration of 2 mu mol/L to be used as a stock solution, the stock solution is subpackaged into 1.5mL of centrifuge tubes, each tube is subpackaged with 50 mu L, and the subpackaged nucleic acid peptide probe solution is placed in a refrigerator at the temperature of-70 ℃ for preservation; before use, 1950. mu.L of ultrapure water is added into the nucleic acid peptide probe stock solution to dilute the solution to 0.05. mu. mol/L, so as to obtain nucleic acid peptide probe working solution which is prepared in situ.
Isotope dilution Mass Spectrometry assay described hereinThe miR-224 content method comprises the following steps of: the synthesized 1mg isotope-labeled polypeptide GDRA [ 2 ]13C6 15N]LFVGEPNR(13C and15n double-labeled leucine L) is ultrasonically dissolved by 300 mu L of acetonitrile, then 700 mu L of ultrapure water is added to prepare isotope labeled polypeptide stock solution with the concentration of about 1mg/mL, the isotope labeled polypeptide stock solution is subpackaged into 1.5mL centrifuge tubes, 100 mu L of isotope labeled polypeptide stock solution is subpackaged into each tube, 900 mu L of ultrapure water is added into each tube to be diluted to about 0.1mg/mL, the isotope labeled polypeptide stock solution is subpackaged into 1.5mL centrifuge tubes, 10 mu L of isotope labeled polypeptide stock solution is subpackaged into each tube, and the subpackaged nucleic acid peptide probe solution is stored in a refrigerator at the temperature of-70 ℃; before use, 990 muL of ultrapure water is added into an isotope labeled polypeptide solution with the concentration of about 0.1mg/mL to be diluted to about 1mg/L, so as to obtain an isotope labeled polypeptide working solution serving as an internal standard, and the isotope labeled polypeptide working solution is prepared as it is and serves as the internal standard in quantification.
The invention discloses a method for determining miR-224 content by isotope dilution mass spectrometry, wherein step (4) is to use a miRNA rapid extraction kit (centrifugal column type) to extract miR-224 in a serum sample, and the method specifically comprises the following steps:
firstly, sucking 0.25ml of serum by using a sample adding gun, adding 0.75ml of lysis solution MRL, and blowing a liquid sample for several times by using the sample adding gun to help to lyse residual cells in the sample;
secondly, shaking and mixing the samples vigorously, and incubating for 5 minutes at 15-30 ℃ to completely decompose the nucleoprotein body;
③ adding 0.15mL of chloroform, tightly covering the sample tube cover, violently shaking for 15 seconds and incubating for 3 minutes at room temperature;
centrifugation at 12,000rpm for 10 minutes at 4 ℃ results in the separation of the sample into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, and RNA exists in the aqueous phase; the volume of the aqueous layer was about 60% of the volume of the added MRL, and the aqueous layer was transferred to a new tube for further processing;
adding 70% ethanol with 0.6 volume, reversing and mixing, and transferring the obtained solution and possible precipitate into an adsorption column RA (the adsorption column is sleeved in a collection tube);
sixthly, centrifuging at 10,000rpm for 45 seconds, collecting the lower filtrate (the lower filtrate contains miRNA), accurately estimating the volume of the lower filtrate, adding 2/3 times of volume of absolute ethyl alcohol, reversing for several times, uniformly mixing, pouring the mixed solution into an adsorption column RB (the volume of the adsorption column RB is about 700 mu L, so that the mixed solution is added into the same adsorption column by centrifuging for several times), centrifuging at 10,000rpm for 30 seconds, and discarding waste liquid;
seventhly, 700 mul of rinsing liquid RW is added, centrifugation is carried out at 12,000rpm for 60 seconds, and waste liquid is discarded;
eighthly, adding 500 mul of rinsing liquid RW, centrifuging at 12,000rpm for 60 seconds, and discarding waste liquid;
ninthly, putting the adsorption column RB back to the empty collection pipe, centrifuging at 12,000rpm for 2 minutes, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;
and (c) taking out the adsorption column RB, putting the adsorption column RB into an RNase free centrifuge tube, adding 30-80 mu l of RNase free water (the heating effect is better in water bath at 65-70 ℃ in advance) in the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 2 minutes, centrifuging at 12,000rpm for 1 minute, and collecting the pure miRNA which is stored at-20 ℃ or lower for later use.
The method for determining miR-224 content by isotope dilution mass spectrometry is disclosed in the invention, wherein the step (5) is to use PierceTMThe RNA 3' End Biotinylation Kit biotinylates miR-224 extracted from a serum sample, and specifically comprises the following steps: dissolving all components in the kit (except 30% of PEG and DMSO) on ice, dissolving DMSO at room temperature, and incubating 30% of PEG at 37 ℃ for 5-10min until the mixture becomes liquid;
adjusting the heater to 85 ℃, and transferring 5 mu of LNon-labeled RNA control and serum miRNA into a microcentrifuge tube; heating at 85 deg.C for 3-5min, and immediately placing on ice;
③ the labeling reaction was carried out, see table 1:
TABLE 1 preparation of RNA ligation reactions
Figure BDA0002118596750000051
Note: the ligation reaction required overnight to improve ligation efficiency.
Adding 70 mu L of nucleic acid free water;
adding 100 mu L chloroform to extract RNA ligase; centrifuging at high speed for 2-3min in a microcentrifuge tube to separate the phases; carefully suck the upper aqueous phase and transfer to nucleic acid-free tubes;
sixthly, 10 mu L of 5M NaCl, 1 mu L of glycogen (glycogen) and 300 mu L of ice-cold 100% ethanol are added, and precipitation is carried out for more than 1h at the temperature of minus 20 ℃;
seventhly, centrifuging for 15min at the temperature of more than 13000g4 ℃, carefully removing supernate without touching precipitates;
washing the precipitate with 300. mu.L of ice-cold 70% ethanol, carefully removing the ethanol, and air-drying the precipitate (5 min);
ninthly, washing the precipitate with ice-cold 70% ethanol, carefully removing the ethanol, and airing the precipitate (5 min);
and (c) resuspending the precipitate in 20 mu L of nucleic acid free water to obtain the biotinylated miRNA (containing miR-224).
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: the streptavidin agarose beads were first reacted with tRNA and BSA (10. mu.L tRNA (10mg/mL) and 10. mu.L BSA (10mg/mL) per 100. mu.L agarose beads), then 20. mu.L of the biotinylated miRNA described above was reacted with 20. mu.L of the treated streptavidin agarose beads, and shaken at 37 ℃ for 2h to form a miRNA-biotin-streptavidin agarose bead complex (comprising miR-224-biotin-streptavidin agarose bead complex).
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: to the above miRNA-biotin-streptavidin agarose ball complex were added 100. mu.L of the nucleic acid peptide probe solution (0.05. mu. mol/L) and 25. mu.L of buffer (10mM Tris, 100mM KCl, 1mM MgCl)2pH 7.4) in a hybridization instrument, and hybridizing at 65 ℃ for 16h to capture the miR-224-biotin-streptavidin agarose ball complex to form the nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball complex. And then, washing the compound with PBS and centrifuging, removing the supernatant, repeating for many times, and removing the unbound nucleic acid peptide probe as much as possible to obtain a pure nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound.
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of:
dissolving the nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound again by 1mL of ultrapure water;
② 10 mu L isotope labeled polypeptide solution (1 mg/L);
③ adding 10 mu L of pancreatin (0.04 mu g/mu L), and standing at 37 ℃ for enzymolysis for 4 hours; since the pancreatin can specifically hydrolyze the peptide bond formed by arginine R and other amino acid hydroxyl groups, the pancreatin can hydrolyze the complex into 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG2-GDR and ALFVGEPNR, and hydrolyze the isotope labeled polypeptide into GDR and A [, ]13C6 15N]LFVGEPNR；
Fourthly, the enzymolysis product is used
Figure BDA0002118596750000061
MAX cartidges (60mg,3mL) solid phase extraction column for purification enrichment ALFVGEPNR and A [, ]13C6 15N]LFVGEPNR；
Figure BDA0002118596750000062
MAX cartridge solid phase extraction column first activated and equilibrated the column with 3mL of methanol and 3mL of ultrapure water, respectively, then the enzymatic product was added, the column was washed with 2mL of ultrapure water, and finally ALFVGEPNR and A were extracted with 1mL of acetonitrile solution containing 0.2% formic acid13C6 15N]LFVGEPNR；
Drying the extract by using nitrogen;
sixthly, 250 mu L of 0.1 percent formic acid aqueous solution is added for redissolving ALFVGEPNR and A after blow drying13C6 15N]LFVGEPNR polypeptide mixtures.
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: using the Shimadzu Nexera X2 high performance liquid chromatography system and AB Sciex 5500 triple quadrupole LC-MS using SymmetryShieldTMRP18 (2.1X 150mm,3.5 μm) column chromatography at 40 deg.CAnd (5) separating. The mobile phase is as follows: a: an aqueous solution containing 0.1% formic acid; b: acetonitrile solution containing 0.1% formic acid; the flow rate is 0.2 mL/min; the sample volume is 10 mu L; elution was performed according to the following gradient: b5% (0-0.1min) → B35% (0.1-6min) → B80% (6-6.1min) → B80% (6.1-8min) → B5% (8-8.1min) → B5% (8.1-12 min). Using a positive ion Multiple Reaction Monitoring (MRM) mode to monitor ALFVGEPNR and A [, ]13C6 15N]Signal strength of two LFVGEPNR polypeptides. The ion source of the mass spectrum system is an electrospray ionization source (ESI), and the spray voltage is 5500V; atomization pressure (Nebulizer pressure)55 psi; the atomization temperature is 550 ℃; atomizing airflow is 10L/min; q1 and Q3 mass selectors each set at unit mass resolution; ions used for quantitative analysis were m/z 501.0 → 571.5(ALFVGEPNR) and m/z 505.5 → 572.2 (A2)13C6 15N]LFVGEPNR); the collision energy CE is 25eV (ALFVGEPNR) and 23eV (A [, ]), respectively13C6 15N]LFVGEPNR); the cluster removing voltage DP is 130V; all data were collected and processed by AB Sciex multisant workstation software (version 3.0.2).
The method for determining miR-224 content by isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of:
the miR-224 stock solution with the concentration of 1 mu mol/L is diluted into the following series of concentrations by DEPC water according to a standard curve: 1000nmol/L, 800nmol/L, 500nmol/L, 300nmol/L, 200nmol/L, 100nmol/L, 80nmol/L, 60nmol/L, 40nmol/L, 20nmol/L, 10nmol/L and 5nmol/L, and then according to the specific steps of the steps (5) - (9), using the Shimadzu Nexera X2 high performance liquid chromatography and AB Sciex 5500 triple four-stage rod LC-MS for extracting the polypeptide product ALFVGEPNR and the isotopically labeled polypeptide A [, ]13C6 15N]Measuring the peak area of LFVGEPNR, establishing a standard curve by taking the concentration of miR-224 as an abscissa (x) and the peak area ratio (y) of the two polypeptides as an ordinate, wherein the curve equation is that y is 0.0103x +0.2147 (R)2＝0.995)；
Collecting serum samples, operating according to the specific steps of (4) - (9), and extracting by adopting Shimadzu Nexera X2 high performance liquid chromatography and AB Sciex 5500 triple four-stage rod LC-MSThe polypeptide product ALFVGEPNR and the isotope labeled polypeptide A13C6 15N]And (3) measuring the peak area of LFVGEPNR, and substituting the peak area ratio of the LFVGEPNR and the peak area ratio of the LFVGEPNR into the curve equation to calculate the content of the miR-224 in the serum sample variety.
Advantageous effects
Compared with the prior art, the method has the following advantages:
the method adopts an isotope dilution mass spectrometry reference technology to directly quantify miR-224 in the serum sample, so that the detection limit is low, and the quantitative result is accurate and reliable; the miR-224 does not need to be amplified in the determination process, so that the influence of amplification efficiency is avoided; the Shimadzu Nexera X2 high performance liquid chromatography system and the AB Sciex 5500 triple quadrupole LC-MS can accommodate about 100 samples for simultaneous detection, and the analysis speed and the analysis flux are significantly better than those of the common method.
Drawings
FIG. 1 nucleic acid peptide Probe 5' -CTAAACGGAACCACTA of the present invention with a concentration of 0.5. mu. mol/L
GTGACTTGA-3' -PEG2-GDRALFVGEPNR and an isotope labeled polypeptide GDRA [ 2 ] with a concentration of 1mg/L13C6 15N]Typical UHPLC-MS/MS chromatograms of LFVGEPNR after being subjected to pancreatin enzymolysis;
FIG. 2 is a standard graph of miR-224;
FIG. 3 is a typical UHPLC-MS/MS chromatogram of a quality control sample;
FIG. 4A serum sample miR-224 and an isotope-labeled polypeptide A [ 2 ]13C6 15N]Typical UHPLC-MS/MS chromatograms of LFVGEPNR after trypsinization.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Instruments and reagents:
AB Sciex 5500 triple quadrupole mass spectrometer (Applied biosystems, USA) equipped with electrospray ion source; the Shimadzu Nexera X2 ultra high performance liquid chromatograph (Japan, Shimadzu corporation) is equipped with a binary high pressure pump, an autosampler and a column oven; ABN2ZA Nitrogen gas generator (PEAK, Inc., USA); SymmetryShieldTMRP18 chromatography column (2.1X 150mm,3.5 μm) (Waters corporation, USA) AllegraTM64R Centrifuge desktop high speed refrigerated Centrifuge (BECKMAN COULTER, USA); an Eppendorf pipettor (Eppendorf, germany); G560E vortex mixer (Scientific Industries, USA); n2Evaporation System NV-15G Nitrogen blower (Agela Technologies, Germany); a hybridization apparatus (china, wuxi voucheri instruments manufacturing ltd); a DKB-501S constant temperature water bath (China, Shanghai sperm macro experimental facilities, Inc.); CSB-10BT ultrasonic cleaner (Hangzhou Aipu Instrument, Inc., China); Milli-Q ultrapure water meter (Merck Millipore, Germany).
Chromatographically pure methanol and acetonitrile were purchased from j.t.baker company (usa); NH (NH)4HCO3And Formic Acid (FA) from Fluke corporation (usa); iodoacetamide (IAM), Dithiothreitol (DTT), urea, PBS available from Sigma-Aldrich, Inc. (USA);
Figure BDA0002118596750000091
MAX cartridges (60mg,3mL) were purchased from Waters corporation (USA); sequence-modified pancreatic enzymes were purchased from Promega (usa); ultrapure water was prepared by the laboratory Milli-Q ultrapure water meter; pierceTMRNA 3' End Biotinylation Kit was purchased from Thermo Fisher Scientific, Inc. (USA); streptavidin agar was purchased from Life Technologies, Inc. (USA); RNase-free BSA, Yeast tRNA from Ambion (USA); nucleic acid peptide probes were synthesized by Hangzhou Taihe Biotechnology, Inc. (China); isotope-labeled polypeptides were synthesized by Shanghai Qiaozhao Biotechnology, Inc. (China); the miR-224 nucleic acid single strand is synthesized by Shanghai Bioengineering Co., Ltd. (China); methodology serum samples for the study experiments were obtained from the medical testing center of the subsidiary hospital of southern university.
The isotope dilution ultra high performance liquid chromatography-mass spectrometry combination method for determining miR-224 in serum comprises the following steps:
(1) preparing a miR-224 nucleic acid single-chain solution, a nucleic acid peptide probe solution and an isotope labeling polypeptide internal standard solution:
each miR-224 nucleic acid single chain tube has a 1OD value, 250 mu L DEPC water is accurately added by a pipette to prepare miR-224 solution with the concentration of 20 mu mol/L and subpackaged into 1.5mL centrifuge tubes, 50 mu L is subpackaged for each tube, then 950 mu L ultrapure water is added into each tube to dilute the solution to the concentration of 1 mu mol/L to be used as stock solution and subpackaged into 1.5mL centrifuge tubes, 20 mu L is subpackaged for each tube, and the subpackaged nucleic acid peptide probe solution is stored in a refrigerator at-70 ℃;
the synthetic amount of the nucleic acid peptide probe is 20nmol, 1mL of ultrapure water is accurately added by a pipette to prepare a nucleic acid peptide probe solution with the concentration of 20 mu mol/L, each tube is subpackaged with 100 mu L, then 900 mu L of ultrapure water is added into each tube to be diluted to the concentration of 2 mu mol/L to be used as a stock solution, the stock solution is subpackaged into 1.5mL of centrifuge tubes, each tube is subpackaged with 50 mu L, and the subpackaged nucleic acid peptide probe solution is placed in a refrigerator at-70 ℃ for storage;
isotope labeled polypeptide GDRA [ 2 ]13C6 15N]The synthesis amount of LFVGEPNR is 1mg, firstly, 300 mu L of acetonitrile is used for ultrasonic dissolution, then 700 mu L of ultrapure water is added to prepare an isotope labeled polypeptide stock solution with the concentration of about 1mg/mL and is subpackaged into 1.5mL centrifuge tubes, 100 mu L of isotope labeled polypeptide stock solution is subpackaged into each tube, 900 mu L of ultrapure water is added into each tube to be diluted to about 0.1mg/mL and is subpackaged into 1.5mL centrifuge tubes, 10 mu L of isotope labeled polypeptide stock solution is subpackaged into each tube, and the subpackaged nucleic acid peptide probe solution is stored in a refrigerator at the temperature of-70 ℃;
a stock solution of miR-224 at a concentration of 1. mu. mol/L was diluted with DEPC water to the following series of concentrations: 1000nmol/L, 800nmol/L, 500nmol/L, 300nmol/L, 200nmol/L, 100nmol/L, 80nmol/L, 60nmol/L, 40nmol/L, 20nmol/L, 10nmol/L, and 5 nmol/L;
diluting the nucleic acid peptide probe stock solution to 0.05 mu mol/L by using ultrapure water as a nucleic acid peptide probe working solution; diluting the isotope labeling polypeptide stock solution to about 1mg/L by using ultrapure water as isotope labeling internal standard polypeptide working solution;
(2) establishing a miR-224 standard curve:
respectively and precisely sucking 5 mu L of miR-224 solution with each concentration according to PierceTMThe RNA 3' End Biotinylation Kit Biotinylation Kit comprises the following steps:
dissolving all components in the kit (except 30% of PEG and DMSO) on ice, dissolving DMSO at room temperature, and incubating 30% of PEG at 37 ℃ for 5-10min until the mixture becomes liquid;
adjusting the heater to 85 ℃, and transferring 5 mu of LNon-labeled RNA control and serum miRNA into a microcentrifuge tube; heating at 85 deg.C for 3-5min, and immediately placing on ice;
③ the labeling reaction was carried out, see table 1:
adding 70 mu L of nucleic acid free water;
adding 100 mu L chloroform to extract RNA ligase; centrifuging at high speed for 2-3min in a microcentrifuge tube to separate the phases; carefully suck the upper aqueous phase and transfer to nucleic acid-free tubes;
sixthly, 10 mu L of 5M NaCl, 1 mu L of glycogen (glycogen) and 300 mu L of ice-cold 100% ethanol are added, and precipitation is carried out for more than 1h at the temperature of minus 20 ℃;
seventhly, centrifuging for 15min at the temperature of more than 13000g4 ℃, carefully removing supernate without touching precipitates;
washing the precipitate with 300. mu.L of ice-cold 70% ethanol, carefully removing the ethanol, and air-drying the precipitate (5 min);
ninthly, washing the precipitate with ice-cold 70% ethanol, carefully removing the ethanol, and airing the precipitate (5 min);
and (c) resuspending the precipitate in 20 mu L of nucleic acid free water to obtain the biotinylated miRNA (containing miR-224).
TABLE 1 preparation of RNA ligation reactions
Figure BDA0002118596750000111
Figure BDA0002118596750000121
Note: the ligation reaction required overnight to improve ligation efficiency.
mu.L of biotinylated miRNA was reacted with 20. mu.L of the treated strepavidin agarose spheres and shaken at 37 ℃ for 2h to form a miRNA-biotin-streptavidin agarose sphere complex (comprising miR-224-biotin-streptavidin agarose sphere complex). Adding 100 mu L of nucleic acid peptide probe solution and 25 mu L of buffer solution into the miRNA-biotin-streptavidin agarose ball compound, placing the mixture into a hybridization instrument for hybridization at 65 ℃ for 16h to capture the miR-224-biotin-streptavidin agarose ball compound to form the nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound, then washing the compound with PBS and centrifuging, removing the supernatant, repeating the steps for a plurality of times, and removing the unbound nucleic acid peptide probe to the greatest extent to obtain the pure nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound. Redissolving the nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound by using 1mL of ultrapure water, adding 10 mu L of isotope labeled polypeptide solution, adding 10 mu L of pancreatin (0.04 mu g/mu L), and placing in a 37 ℃ constant temperature water bath box for enzymolysis for 4 h. Subjecting the enzymolysis product to
Figure BDA0002118596750000122
MAX cartidges (60mg,3mL) solid phase extraction column for purification enrichment ALFVGEPNR and A [, ]13C6 15N]LFVGEPNR；
Figure BDA0002118596750000123
MAX cartridge solid phase extraction column first activated and equilibrated the column with 3mL of methanol and 3mL of ultrapure water, respectively, then the enzymatic product was added, the column was washed with 2mL of ultrapure water, and finally ALFVGEPNR and A were extracted with 1mL of acetonitrile solution containing 0.2% formic acid13C6 15N]LFVGEPNR; drying the extract by using nitrogen; after blow drying, 250 μ L of 0.1% formic acid aqueous solution is added to redissolve ALFVGEPNR and A13C6 15N]LFVGEPNR polypeptide mixtures.
Under the same preset condition of the ultra performance liquid chromatography mass spectrum, respectively injecting a series of redissolved polypeptide and isotope labeled polypeptide samples with different known concentrations, of which the volume is 10 mu L, into an ultra performance liquid chromatography-mass spectrum combination instrument to obtain a series of chromatograms of miR-224 standard curve working solutions with different known concentrations;
the polypeptide ALFVGEPNR after redissolution and the isotope labeled polypeptide A [, ]13C6 15N]Taking the ratio of the peak areas of the LFVGEPNR chromatographic peak as a vertical coordinate and taking the concentration of the working solution of the corresponding miR-224 standard curve as a horizontal coordinate to establish a miR-224 standard curve;
chromatographic conditions are as follows:
a chromatographic column: SymmetryShieldTMRP18 (2.1X 150mm,3.5 μm) chromatography column; mobile phase: a: an aqueous solution containing 0.1% formic acid (vol./vol.); b: acetonitrile solution containing 0.1% formic acid, gradient elution, elution procedure: b5% (0-0.1min) → B35% (0.1-6min) → B80% (6-6.1min) → B80% (6.1-8min) → B5% (8-8.1min) → B5% (8.1-12 min); flow rate: 0.2 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; mass spectrum conditions:
an ion source: electrospray ion source (ESI); scanning mode: a positive ion mode; capillary/spray voltage: 5500V; ion source atomization temperature: 550 ℃; atomizing airflow: 10L/min; ion source atomization pressure (Nebulizer pressure): 55 psi; heating auxiliary gas by an ion source: 60 psi; air curtain air: 30 psi; collision gas: 4 psi; the gas is nitrogen; scanning mode: multiple reaction monitoring, the multiple reaction monitoring conditions are shown in table 2 below:
TABLE 2 ALFVGEPNR and A [ ]13C6 15N]Multiple reaction monitoring conditions for LFVGEPNR
Figure BDA0002118596750000131
All data were collected and processed by AB Sciex multisant workstation software (version 3.0.2).
(3) Collecting a chromatogram of the serum sample solution:
extracting miRNA in a serum sample by using an miRNA rapid extraction kit (a centrifugal column type), then precisely absorbing 5 mu L of miRNA sample, and carrying out an experiment according to a biotinylation process for establishing an miRNA-224 standard curve;
under the same preset condition of the ultra performance liquid chromatography mass spectrum in the step (2), injecting the sample solution with the volume of 10 mu L after extraction, blow-drying and redissolving into an ultra performance liquid chromatography mass spectrum combination instrument to obtain a chromatogram of the serum sample solution;
(4) determination of miR-224 concentration in serum samples:
subjecting the polypeptide ALFVGEPNR and the isotope labeled polypeptide A in the chromatogram of the serum sample solution13C6 15N]Substituting the ratio of the peak areas of the LFVGEPNR chromatographic peaks into the established miR-224 standard curve equation, and calculating the concentration of miR-224 in the serum sample;
(5) collecting a chromatogram of a quality control sample and a sample solution for standard addition recovery:
precisely sucking 5 mu L of miR-224 quality control product (300nmol/L), and performing an experiment according to a biotinylation process for establishing a miRNA-224 standard curve;
extracting miRNA (duplicate) in a serum sample with known miR-224 concentration by using an miRNA rapid extraction kit (centrifugal column type), wherein the final volume is 20 mu L (tube 1 and tube 2), adding miR-224 standard solutions (5 mu L) with different concentrations into each tube, fully mixing the solutions, precisely absorbing 5 mu L of standard samples in each tube, and performing experiments according to a biotinylation process for establishing an miRNA-224 standard curve;
under the same preset condition of the ultra performance liquid chromatography mass spectrum in the step (2), injecting the sample solution with the volume of 10 mu L after extraction, drying and redissolving into an ultra performance liquid chromatography mass spectrum combination instrument to obtain chromatograms of the quality control product and the standard sample solution;
the polypeptide ALFVGEPNR and the isotope labeled polypeptide A in the chromatograms of the quality control product and the labeled sample solution13C6 15N]Substituting the ratio of the peak areas of the LFVGEPNR chromatographic peak into the established miR-224 standard curve equation, and calculating the concentration of miR-224 in the quality control product and the standard sample;
(6) and (3) calculating the standard addition recovery rate:
Figure BDA0002118596750000151
(7) linear relationship, lowest detection limit and lowest quantitation limit:
precisely absorbing 10 mu L of miR-224 standard curve working solution with different concentrations under the same ultra performance liquid chromatography mass spectrum condition in the step (2), respectively injecting the working solution into an ultra performance liquid chromatograph, and carrying out parallel analysis for 3 times for each concentration; recording the polypeptide ALFVGEPNR and the isotope labeled polypeptide A in the working solution of each concentration13C6 15N]The peak area of LFVGEPNR (as shown in FIG. 1, FIG. 1 is a nucleic acid peptide probe 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG2-GDRALFVGEPNR at a concentration of 0.5. mu. mol/L and an isotopically labeled polypeptide GDRA [ 2 ] at a concentration of 1mg/L13C6 15N]LFVGEPNR is subjected to enzymolysis by trypsin to obtain ALFVGEPNR and A [ [ alpha ] ]13C6 15N]LFVGEPNR typical UHPLC-MS/MS chromatogram) with the polypeptide ALFVGEPNR and the isotope labeled polypeptide A [ 2 ]13C6 15N]Taking the ratio of peak areas of LFVGEPNR chromatographic peaks as a vertical coordinate, taking the miR-224 concentration of a corresponding miR-224 standard curve working solution as a horizontal coordinate to perform linear regression, wherein the regression equation y is 0.0103x +0.2147, and the correlation coefficient R2Fig. 2 shows that the method of the present embodiment has a good linear relationship within a preset linear range;
the sensitivity of the established method is examined 6 times in parallel by analyzing each concentration sample by taking the linear lowest point concentration (0.5nmol/L) as the lowest limit of quantitation (LLOQ) and the concentration (0.25nmol/L) which is lower than the concentration (0.5nmol/L) which is two times that of the LLOQ as the lowest limit of detection (LLOD), and the result shows that the accuracy of the LLOD detection result of the established method is 105.9%, the Relative Standard Deviation (RSD) is 9.56%, the accuracy of the LLOQ detection result is 94.5% and the RSD is 7.84%, which shows that the sensitivity of the established method is high and the accuracy of low-concentration detection is high;
(9) and (3) precision test:
preparing low-concentration (42.8nmol/L), medium-concentration (236.4nmol/L) and high-concentration (856.3nmol/L) quality control samples according to a quality control sample processing method, respectively marking the quality control samples as QCL, QCM and QCH, continuously detecting each concentration quality control sample for 3 times as the intra-day precision, and continuously detecting for 5 days as the inter-day precision; as a result: the RSD of the miR-224 in the quality control samples with different concentrations is less than 5.86% in-day and in-day precision, which indicates that the method established in the embodiment has good precision, and the results are shown in Table 3;
(10) and (3) stability test:
according to quality control samples QCL, QCM and QCH: 1) placing the blood sample in an autosampler at 4 ℃, measuring for 0, 2, 4, 6, 8 and 12 hours, and inspecting the stability of miR-224 in the blood sample when the autosampler is stored for 12 hours; 2) after the mixture is placed at room temperature for 2 hours, the stability of miR-224 in a blood sample after being placed at room temperature for 2 hours is inspected; 3) placing the blood sample in a refrigerator at the temperature of-70 ℃, repeatedly freezing and thawing for 1 time and 3 times, inspecting the stability of miR-224 in the blood sample in the repeated freezing and thawing process, and measuring the quality control samples with different concentrations under the same ultra performance liquid chromatography mass spectrometry condition as that in the step (2), wherein each concentration quality control sample is prepared in 6 parts in parallel; as a result: RSD of miR-224 stability in quality control samples with different concentrations is less than 3.25%, which indicates that miR-224 stability in blood samples is good, and the results are shown in Table 3;
table 3 precision and stability test results of miR-224 (RSD ═ 6)
Figure BDA0002118596750000161
(11) And (3) standard recovery rate test:
adding a miR-224 standard substance into a serum sample with known miR-224 concentration of 105.7nmol/L, respectively preparing low-concentration, medium-concentration and high-concentration standard recovery samples according to a standard recovery sample treatment method, respectively marking the samples as RL, RM and RH, parallelly preparing 6 parts of each concentration sample, and inspecting the accuracy of miR-224 in the blood sample measured by the method established in the embodiment; as a result: the average recovery rate of the samples with different concentration standard recovery rates is 99.6-104.6%, and the RSD is less than 7.04%, which indicates that the method established by the embodiment has good accuracy, and the results are shown in Table 4; typical UPLC-MS/MS chromatograms of the obtained quality control sample and spiked recovery sample solutions are shown in fig. 3;
table 4 miR-224 normalized recovery results (n ═ 6)
Figure BDA0002118596750000171
Figure BDA0002118596750000181
Determination of miR-224 in serum samples:
preparing a blood sample according to a serum sample treatment method, precisely sucking 10 mu L of the blood sample under the same ultra performance liquid chromatography mass spectrum condition as that in the step (2), and respectively injecting the blood sample into an ultra performance liquid chromatography to obtain a serum sample chromatogram, wherein the chromatogram is shown in figure 4; the results are shown in Table 5;
TABLE 5 serum sample miR-224 assay results (nmol/L)
Figure BDA0002118596750000182
Figure BDA0002118596750000191
The results show that the isotope dilution ultra liquid chromatography-mass spectrometry combined method for measuring miR-224 in serum, which is constructed in the embodiment, is used for measuring miR-224 in serum, and has the advantages of simplicity, rapidness, high accuracy, strong specificity, high precision and good sensitivity.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (8)

1. A method for determining miR-224 content by isotope dilution mass spectrometry comprises the following steps:
(1) preparing the synthesized miR-224 nucleic acid single strands into a solution for establishing a standard curve;
(2) synthesizing a nucleic acid peptide probe to prepare a solution for capturing miR-224;
(3) preparing the synthesized isotope-labeled polypeptide into solution as an internal standard;
(4) extracting miRNA from a serum sample;
(5) biotinylating the extracted miRNA;
(6) reacting the biotinylated miRNA-224 with streptomycin agarose spheres to form a miRNA-224-biotin-streptavidin agarose sphere compound;
(7) adding a nucleic acid peptide probe to capture a miR-224-biotin-streptavidin agarose ball compound;
(8) cleaning a nucleic acid peptide probe-miR-224-biotin-streptavidin agarose bead compound, adding trypsin for enzymolysis, performing solid phase extraction on an enzymolysis product, drying and redissolving extracted polypeptide;
(9) carrying out mass spectrometry on the redissolved polypeptide and the isotope-labeled polypeptide sample;
(10) calculating the content of miR-224 in the serum sample;
the step (8) specifically comprises the following steps:
dissolving the nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound again by using 1mL of ultrapure water;
adding 10 microliter of 1mg/L isotope labeled polypeptide solution;
③ adding 10 mu L of 0.04 mu g/mu L pancreatin, and standing at 37 ℃ for enzymolysis for 4 hours;
fourthly, 60mg of 3mL of Oasis is used for the enzymolysis product®The MAX cartidges solid phase extraction column is purified and enriched with ALFVGEPNR and A [ ]13C6 15N]LFVGEPNR；Oasis®MAX cartridge solid phase extraction column first activated and equilibrated the column with 3mL of methanol and 3mL of ultrapure water, respectively, then the enzymatic product was added, the column was washed with 2mL of ultrapure water, and finally ALFVGEPNR and A were extracted with 1mL of acetonitrile solution containing 0.2% formic acid13C6 15N]LFVGEPNR；
Drying the extract with nitrogen;
sixthly, after blow drying, adding 250 mu L of 0.1 percent formic acid aqueous solution to redissolve ALFVGEPNR and A [13C615N ] LFVGEPNR polypeptide mixture;
the step (9) specifically comprises the following steps: using the Shimadzu Nexera X2 high Performance liquid chromatography System and AB Sciex 5500 triple quadrupole LC Mass spectrometer, 60mg,3mL of Symmetry Shield was usedTMPerforming chromatographic separation on an RP18 chromatographic column at 40 ℃; the mobile phase is as follows: a: an aqueous solution containing 0.1% formic acid; b: acetonitrile solution containing 0.1% formic acid; the flow rate is 0.2 mL/min; the sample volume is 10 mu L; elution was performed according to the following gradient: b5%, 0-0.1min → B35%, 0.1-6min → B80%, 6-6.1min → B80%, 6.1-8min → B5%, 8-8.1min → B5%, 8.1-12min → B5%; using a positive ion multi-reaction monitoring mode to monitor ALFVGEPNR and A13C6 15N]Signal strength of two LFVGEPNR polypeptides; the ion source of the mass spectrum system is an electrospray ionization source, and the spraying voltage is 5500V; atomization pressure 55 psi; the atomization temperature is 550 ℃; atomizing airflow is 10L/min; q1 and Q3 mass selectors each set at unit mass resolution; ions used for quantitative analysis were m/z 501.0 → 571.5 and m/z 505.5 → 572.2, respectively; the collision energy CE is 25eV and 23eV, respectively; the cluster removing voltage DP is 130V; all data were collected and processed by AB sciex multiquant workstation software;
the step (10) specifically comprises the following steps:
establishing a standard curve, and diluting miR-224 stock solution with the concentration of 1 mu mol/L into the following series of concentrations by using DEPC water: 1000nmol/L, 800nmol/L, 500nmol/L, 300nmol/L, 200nmol/L, 100nmol/L, 80nmol/L, 60nmol/L, 40nmol/L, 20nmol/L, 10nmol/L and 5nmol/L, and then according to the specific steps of the steps (5) - (9), using the Shimadzu Nexera X2 high performance liquid chromatography and AB Sciex 5500 triple four-stage rod LC-MS for extracting the polypeptide product ALFVGEPNR and the isotopically labeled polypeptide A [, ]13C6 15N]The peak area of LFVGEPNR is measured, and the concentration of miR-224 is taken as the abscissa (x) The peak area ratio of the two polypeptides (A), (B), (C)y) Establishing a standard curve for the ordinate, the equation of the curve beingy=0.0103x+0.2147，R 2=0.995；
Collecting serum sample, operating according to the specific steps of (4) - (9), and producing ALFVGEPNR and isotope labeled polypeptide A [ 2 ] from the extracted polypeptide by using Shimadzura X2 high performance liquid chromatography and AB Sciex 5500 triple four-stage rod LC MS13C6 15N]And (3) measuring the peak area of LFVGEPNR, and substituting the peak area ratio of the LFVGEPNR and the peak area ratio of the LFVGEPNR into the curve equation to calculate the miR-224 content in the serum sample.
2. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (1) specifically comprises the following steps: the synthetic amount of the miR-224 nucleic acid single strand is 4OD value, the 1OD value of each tube is subpackaged, 250 mu L DEPC water is accurately added into each tube to prepare miR-224 solution with the concentration of 20 mu mol/L and subpackaged into 1.5mL centrifuge tubes, 50 mu L of each tube is subpackaged, then 950 mu L of ultrapure water is added into each tube to be diluted to the concentration of 1 mu mol/L to be used as stock solution and subpackaged into 1.5mL centrifuge tubes, 20 mu L of each tube is subpackaged, and the subpackaged nucleic acid peptide probe solution is placed in a refrigerator at-70 ℃ for storage; before use, DEPC water was added to the miR-224 stock solution to dilute the same into a series of concentrations for establishing a standard curve.
3. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (2) specifically comprises the steps of:
the nucleic acid peptide probe sequence is 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG2-GDRALFVGEPNR,
wherein 5'-CTAAACGGAACCACTAGTGACTTGA-3' is a probe nucleic acid portion that is complementary paired to miR-224 sequence 5'-UCAAGUCACUAGUGGUUCCGUUUAG-3'; GDRALFVGEPNR is a probe peptide portion characterized by a non-human any protein pancreatin hydrolyzed specific peptide segment comprising a pancreatin hydrolysis site arginine R; PEG2 links nucleic acids to peptides; the synthesis amount of the nucleic acid peptide probe is 20nmol, 1mL of ultrapure water is accurately added to prepare a nucleic acid peptide probe solution with the concentration of 20 mu mol/L, each tube is subpackaged with 100 mu L, then 900 mu L of ultrapure water is added into each tube to be diluted to the concentration of 2 mu mol/L to be used as a stock solution, the stock solution is subpackaged into 1.5mL of centrifuge tubes, each tube is subpackaged with 50 mu L, and the subpackaged nucleic acid peptide probe solution is placed in a refrigerator at the temperature of-70 ℃ for preservation; before use, 1950. mu.L of ultrapure water is added into the nucleic acid peptide probe stock solution to dilute the solution to 0.05. mu. mol/L, so as to obtain nucleic acid peptide probe working solution which is prepared in situ.
4. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (3) specifically comprises the steps of: the synthesized 1mg isotope-labeled polypeptide GDRA [ 2 ]13C6 15N]LFVGEPNR is firstly ultrasonically dissolved by 300 mu L of acetonitrile, then 700 mu L of ultrapure water is added to prepare an isotope labeled polypeptide stock solution with the concentration of about 1mg/mL, the isotope labeled polypeptide stock solution is subpackaged into 1.5mL centrifuge tubes, 100 mu L of ultrapure water is subpackaged into each tube, 900 mu L of ultrapure water is added into each tube to be diluted to about 0.1mg/mL and is subpackaged into 1.5mL centrifuge tubes, 10 mu L of ultrapure water is subpackaged into each tube, and the subpackaged nucleic acid peptide probe solution is stored in a refrigerator at the temperature of-70 ℃; before use, 990 muL of ultrapure water is added into an isotope labeled polypeptide solution with the concentration of about 0.1mg/mL to be diluted to about 1mg/L, so as to obtain an isotope labeled polypeptide working solution serving as an internal standard, and the isotope labeled polypeptide working solution is prepared as it is and serves as the internal standard in quantification.
5. The method for determining the content of miR-224 by using isotope dilution mass spectrometry as claimed in claim 1, wherein the step (4) is to use a miRNA rapid extraction kit to extract miR-224 from a serum sample, and the method specifically comprises the following steps:
firstly, sucking 0.25mL of serum by using a sample adding gun, adding 0.75mL of lysis solution MRL, and blowing a liquid sample for several times by using the sample adding gun to help to lyse residual cells in the sample;
secondly, shaking and mixing the samples vigorously, and incubating for 5 minutes at 15-30 ℃ to completely decompose the nucleoprotein body;
③ adding 0.15mL of chloroform, tightly covering the sample tube cover, violently shaking for 15 seconds and incubating for 3 minutes at room temperature;
centrifugation at 12,000rpm for 10 minutes at 4 ℃ results in the separation of the sample into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, and RNA exists in the aqueous phase; the volume of the aqueous layer was about 60% of the volume of the added MRL, and the aqueous layer was transferred to a new tube for further processing;
adding 0.6 volume of 70% ethanol, reversing and mixing uniformly, and transferring the obtained solution and possible precipitates into an adsorption column RA, wherein the adsorption column is sleeved in a collection tube;
sixthly, centrifuging at 10,000rpm for 45 seconds, collecting lower filtrate, accurately estimating the volume of the lower filtrate, adding 2/3 times of anhydrous ethanol, reversing for several times, uniformly mixing, pouring the mixed solution into an adsorption column RB, centrifuging at 10,000rpm for 30 seconds, and discarding waste liquid;
seventhly, 700 mu L of rinsing liquid RW is added, centrifugation is carried out at 12,000rpm for 60 seconds, and waste liquid is discarded;
eighthly, adding 500 mu L of rinsing liquid RW, centrifuging at 12,000rpm for 60 seconds, and discarding waste liquid;
ninthly, putting the adsorption column RB back to the empty collection pipe, centrifuging at 12,000rpm for 2 minutes, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;
and removing the adsorption column RB from the chloride, putting the adsorption column RB into an RNase free centrifuge tube, adding 30-80 mu L of RNase free water in the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 2 minutes, centrifuging at 12,000rpm for 1 minute, collecting the pure miRNA, and storing at-20 ℃ or lower for later use.
6. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (5) is to use PierceTMThe RNA 3' End Biotinylation Kit biotinylates miR-224 extracted from a serum sample, and specifically comprises the following steps:
dissolving all other components in the kit on ice except 30% of PEG and DMSO, dissolving the DMSO at room temperature, and incubating the 30% of PEG at 37 ℃ for 5-10min until the mixture becomes liquid;
adjusting a heater to 85 ℃, and transferring 5 mu L of Non-labeled RNA control and serum miRNA into a microcentrifuge tube; heating at 85 deg.C for 3-5min, and immediately placing on ice;
carrying out a marking reaction:
adding 70 mu L of nucleic acid free water;
adding 100 mu L chloroform to extract RNA ligase; centrifuging at high speed for 2-3min in a microcentrifuge tube to separate the phases; carefully suck the upper aqueous phase and transfer to nucleic acid-free tubes;
sixthly, 10 mu L of 5M NaCl, 1 mu L of glycogen and 300 mu L of ice-cold 100% ethanol are added, and the mixture is precipitated for more than 1h at the temperature of minus 20 ℃;
seventhly, centrifuging for 15min at the temperature of 4 ℃ of more than 13000g, and carefully removing supernatant without touching sediment;
washing the precipitate with 300. mu.L of ice-cold 70% ethanol, carefully removing the ethanol, and air-drying the precipitate;
ninthly, washing the precipitate with ice-cold 70% ethanol, carefully removing the ethanol, and airing the precipitate;
and (c) resuspending the precipitate in 20 mu L of nucleic acid free water to obtain the biotinylated miRNA, wherein the biotinylated miRNA is contained in miR-224.
7. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (6) specifically comprises the steps of: reacting streptomycin agarose spheres with tRNA and BSA, then reacting 20. mu.L of biotinylated miRNA in step (5) with 20. mu.L of treated streptomycin agarose spheres, shaking the mixture at 37 ℃ for 2h to form a miRNA-biotin-streptavidin agarose sphere complex comprising miR-224-biotin-streptavidin agarose sphere complex.
8. The method for determining miR-224 content by isotope dilution mass spectrometry according to claim 1, wherein the step (7) specifically comprises the steps of: adding 100 mu L of 0.05 mu mol/L nucleic acid peptide probe solution and 25 mu L of buffer solution into the miRNA-biotin-streptavidin agarose ball compound obtained in the step (6), placing the mixture into a hybridization instrument for hybridization at 65 ℃ for 16h to capture the miR-224-biotin-streptavidin agarose ball compound, and forming the nucleic acid peptide probe-miR-224-biotin-streptavidin-agarose ball compound; then, washing the compound with PBS and centrifuging, removing the supernatant, repeating for many times, and removing the unbound nucleic acid peptide probe to obtain a pure nucleic acid peptide probe-miR-224-biotin-streptavidin agarose ball compound;
wherein the buffer solution is 10mM Tris, 100mM KCl, 1mM MgCl2，pH 7.4。
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