CN106048037A - Human circulating miR-122 detection method - Google Patents
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Abstract
The invention relates to a human circulating miR-122 detection method. The method includes: extracting serum total RNA (ribonucleic acid) as a sample, dissolving the sample into distilled water without nuclease, taking part of solution to perform reverse transcription to obtain cDNA, carrying out miR-122 specificity real-time fluorescent quantitative PCR (polymerase chain reaction) to obtain Ct values, and verifying chip reflected results. The human circulating miR-122 detection method has advantages that sensitivity is improved, a new clinical basis is provided for human circulating miR-122 variation under a condition of obesity, and circulating miR-122 level is closely related to obesity, glycoregulation and insulin resistance.
Description
Technical field
The invention belongs to obesity diagnosis biological markers field, circulate the detection method of miR-122 particularly to a kind of people.
Background technology
Research shows, adipose cell plays central role in the pathogenic process of insulin resistant.Body is in fat state
Under, adipose cell secretory function is disorderly, lipid excessive buildup, and then causes the pathological processes such as cytolipin toxicity, insulin resistant
Generation.Basic research currently, with respect to obesity tentatively discloses its pathogenesis, but can be used for obesity and diagnose biology
Mark therapeutic effect the deficientest, existing is the most not fully up to expectations.
Recently basic research finds, miRNAs plays important work in obesity generation, insulin resistant generation evolution
With, but its regulatory mechanism not yet illustrates.In tumor, cardiovascular disease and metabolic disease field, specific miRNAs has become latent
At biological markers and pharmaceutical intervention target spot.2013, medicine MRX34 prepared by transformation miR-34 molecular structure had been enter into I
Clinical trial phase, target group is to be unsuitable for the primary hepatocarcinoma of excision or involve the solid tumor patient of liver.
Endogenous circulation miRNAs has relatively stable, Wicresoft, is prone to the features such as detection, under morbid state its express spectra and
Expression significantly changes, and is expected to be used for medical diagnosis on disease, pathological, prognosis evaluation.But, existing screening particular cycle miRNAs
Cohort study's sample size as obesity diagnosis and treatment biological markers is little, and the result of different institute reports is not consistent.Recently
Research finds, blood plasma miR-142-3p, miR-140-5p, miR-423-5p, miR-520c-3p and miR-15a are probably obesity wind
Danger assessment and the new biological markers of classification of diseases.Another studies prompting, serum miR-15b, miR-138, miR-376a
Can be used as predicting effective biological indicator of obesity.More clinical evidence is needed badly with the relation of Metabolism regulation about circulation miRNAs.
Summary of the invention
The technical problem to be solved is to provide a kind of people and circulates the detection method of miR-122, and the method detects
Method improves sensitivity, provides new clinic foundation for circulating the change of miRNAs under body fat state, circulates miR-
122 levels and obesity, sugar regulate, insulin resistant is closely related；Particular, it is important that present invention demonstrates circulation miR-122
Level rise is the independent hazard factor of insulin resistant；Circulation miR-122 has as diagnosis and treatment obesity, insulin resistant
The bright prospects of serologic marker thing.
A kind of people of the present invention circulates the detection method of miR-122, including:
Sample, as sample, is dissolved in the distilled water of nuclease, takes Partial Inverse and be transcribed into cDNA by extraction serum total serum IgE,
Carry out the reaction of miR-122 specificity real-time fluorescence quantitative PCR, obtain the result that chip reflects by Ct value and verify,.
Consisting of of the reverse transcription mixed liquor that described reverse transcription uses: 5 × reverse transcription reaction liquid 2 μ l, reverse transcriptase mix 1
μ l, Quality Control Spike-In solution 0.5 μ l, remove the distilled water 4.5 μ l of nuclease.
Described reverse transcription condition is: 42 DEG C of reaction 1h, and 95 DEG C of reaction 5min are subsequently reduced to 4 DEG C.
The reaction system of described real-time fluorescence quantitative PCR is: cDNA template 4 μ l, primer 1 μ l, 2 × SYBR 5 μ l.
The reaction condition of described real-time fluorescence quantitative PCR is: 95 DEG C of denaturations 10min；95 DEG C of 10s, 60 DEG C extend 1min,
45 circulations.
Beneficial effect
Detection method improves sensitivity, provides new for circulating the change of miRNAs under body fat state
Clinical foundation, circulation miR-122 level and obesity, sugar regulate, insulin resistant is closely related；Particular, it is important that the present invention
Confirm that circulation miR-122 level rise is the independent hazard factor of insulin resistant；Circulation miR-122 has as diagnosis and treatment fertile
Fat disease, the bright prospects of serologic marker thing of insulin resistant.
Accompanying drawing explanation
Fig. 1 (A) is the comparison of normal type matched group (n=107) and obesity patient's group (n=123) circulation miR-122；
(B) it is that normal type matched group organizes circulation miR-122 realtime fluorescent quantitative PCR experiment original Ct value with obesity patient；(C) it is
After correction sex, age, ALT, HDL-c, obesity patient organizes the comparison of circulation miR-122 level and normal type matched group；
Fig. 2 is obesity patient's serum miR-122 level in various degree；Wherein, (A) be Mild Obesity group (n=28), in
The fat group (n=66) of degree, severe simple obesity group (n=29) circulation miR-122 and the comparison of normal type matched group (n=107)；
(B) after for correction sex, age, ALT, HDL-c, Mild Obesity group, central obesity group, severe simple obesity group circulation miR-122 water
The flat comparison with normal type matched group.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and
It is not used in restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
Study population: 18～30 years old crowd.Obesity patient is the patient of fat disease that calls for specialized treatment outpatient service；Normal type experimenter
It is students volunteer.
1. serum RNA extracting
The extraction of serum total serum IgE uses microRNA extraction agent box (miRNeasy Mini Kit), and with reference to Germany
The description that Qiagen company provides.
(1) serum sample is taken out in-80 DEG C of refrigerators, be placed in and melt the most completely；
(2) 1.5ml QIAzol lysate is added to each equipped with in the EP pipe of serum sample, to mix at whirlpool the most at a high speed
In clutch, acutely vibration 15s is placed on ice, this process repeatable, until white precipitate is wholly absent, room temperature stands 5min；
(3) often pipe adds 200 μ l chloroformic solutions, with the 15s that acutely vibrates on eddy mixer the most at a high speed, stands 3min；
(4) 4 DEG C, 12,000g are centrifuged 15min, by the EP pipe of colourless for upper strata water sample solution to another RNAse Free, add
Softly mix after entering 1.5 times of volume dehydrated alcohol；
(5) drawing 700 μ l mixture and add RNeasy Mini centrifugal column, room temperature 9000g is centrifuged 15s, abandons 2ml collecting pipe
In liquid, repeat this step until all mixture all add RNeasy Mini centrifugal column；
(6) adding 700 μ l RWT washing liquids to each RNeasy Mini centrifugal column, room temperature 9000g is centrifuged 15s, abandons 2ml and receives
Liquid in collector；
(7) adding 500 μ l RPE washing liquids to each RNeasy Mini centrifugal column, room temperature 9000g is centrifuged 15s, abandons 2ml and receives
Liquid in collector；
(8) adding 500 μ l RPE washing liquids to each RNeasy Mini centrifugal column again, room temperature 9000g is centrifuged 2min, abandons 2ml
Collecting pipe；
(9) being placed on new 2ml collecting pipe by RNeasy Mini centrifugal column, room temperature maximum (top) speed is centrifuged 1min, to remove
The dehydrated alcohol of centrifugal column remaining；
(10) RNeasy Mini centrifugal column is placed on the 1.5ml centrifuge tube of RNAse Free, to each centrifugal column filter membrane
Central authorities add the distilled water of 30 μ l RNAse Free, and room temperature 12,000g is centrifuged 1min eluted rna；
(11) room temperature maximum (top) speed is centrifuged 1min, the RNA on abundant eluting filter membrane, and RNA is transferred to new RNAse
In the centrifuge tube of Free.
2. serum miRNAs Microarray chip
(1) it is the impact getting rid of sample room individual variation as far as possible, it may be found that the serum miRNAs of crowd obesity group patient
Sample is mixed into 2 biased samples (pooled sample), each fat group biased sample serum containing 28 patients at random
MiRNAs sample, regular restructuring is mixed into 2 biased samples the most at random；
(2) according to the biased sample concentration recorded by Aglient 2100 biological analyser, the RNA sample that total amount is 100ng is taken
Product, put into 1.5ml centrifuge tube, are placed on ice；
(3) add 2 μ l calf intestinal phosphatase esterase (calf intestinal phosphatase, CIP) mix, softly mix
Even, 37 DEG C of water-bath 30min, so that sample dephosphorylation, CIP mix matching while using, it is placed on ice, table specific as follows:
| Reagent | Volume |
| 10 × CIP buffer | 0.4μl |
| CIP | 0.5μl |
| Quality Control Spike-In solution | 1.1μl |
| Amount to | 2μl |
The amount of polygamy 10% is standby every time；
(4) 2.8 μ l DMSO, 100 DEG C of water-bath 5～10min degeneration are added；
(5) being transferred on ice by sample immediately, each sample adds 4.5 μ l ligase mix, softly mixes, 16 DEG C of water-baths
2h, T4RNA ligase mix matching while using, it is placed on ice, table specific as follows:
| Reagent | Volume |
| 10 × T4RNA ligase buffer | 1μl |
| Cyanine3-pCp | 3μl |
| T4RNA ligase | 0.5μl |
| Amount to | 4.5μl |
The amount of polygamy 10% is standby every time；
(6) 50 DEG C are concentrated in vacuo 30min～1h, until sample is completely dried；
(7) with the resuspended sample of distilled water of 17 μ l RNAse Free, often pipe adds 28 μ l and hybridizes mix (each polygamy 10%
Amount standby), as shown in the table:
| Reagent | Volume |
| 10 × gene blockage liquid (gene expression blocking agent) | 4.5μl |
| Quality Control Spike-In solution | 1μl |
| 2 × Hi-RPM hybridization solution | 22.5μl |
| Amount to | 28μl |
Total system is 45 μ l, fully mixes；
(8) 100 DEG C of water-bath 5min, are immediately transferred into ice bath 5min；
(9) by sample and miRNA (8*70K) V16.0 chip in rolling hybrid heater 55 DEG C, 20rpm rolls hybridization 20h；
(10) after having hybridized, chip is placed in and washes in cylinder, with the gene detergent (GE adding 10%Triton X-102
Wash Buffer) 1, gene detergent 2 develops a film 5-6 time；
(11) chip results uses Aglient gene chip scanning instrument to be scanned, soft with Feature Extraction
Part 10.7 reads result, arranges scanning resolution=5 μm, PMT100%, 5%；
(12) using Gene Spring software 11.0 all data to be normalized, algorithm used is quantile
Method (Quantile).
The reverse transcription of 3.miRNAs
The reverse transcription of miRNAs uses the general Reverse Transcriptase kit of miRCURY LNA (miRCURY LNA Universal
RT Kit), and the description provided with reference to Exiqon company of Denmark.
(1) from 30 μ l RNA solution, accurately draw 2 μ l RNA solution to be placed in the EP pipe of new RNAse Free, be placed on ice；
(2) each sample of description provided according to Exiqon company prepares reverse transcription mix mixed enzyme by following system
Solution, specific as follows:
| Reagent | Volume |
| 5 × reverse transcription reaction liquid (reaction buffer) | 2μl |
| Reverse transcriptase mix | 1μl |
| Quality Control Spike-In solution | 0.5μl |
| Remove the distilled water of nuclease | 4.5μl |
| Total | 8μl |
The amount of polygamy 10% is standby every time；
(3) add 8 μ l mix mixed solutions to each sample, be quickly centrifuged, put into PCR instrument 42 DEG C reaction 1h, 95 DEG C
5min, is down to 4 DEG C immediately；
(4) adding 390 μ l in cDNA product goes the distilled water diluting of nuclease to 400 μ l standby (40 times of dilutions).
4. real-time quantitative PCR (real-time PCR)
Use real-time fluorescence quantitative PCR test kit and people's miR-122 specific primer (Exiqon Products), with reference to red
The description that wheat Exiqon company provides, carries out real-time quantitative PCR, each sample on Roche LightCycler 480 instrument
3 multiple holes are set.
(1) reaction system
| Reagent | Volume |
| Dilute cDNA template | 4μl |
| People's miR-122PCR primer | 1μl |
| 2×SYBR | 5μl |
| Total | 10μl |
(2) reaction condition
Stage 1 denaturation: 95 DEG C of 10min
Stage 2 expands: 95 DEG C of 10s, 60 DEG C of 1min (1.6 DEG C/s), 45Cycles；
Stage 3 solubility curve analysis.
(3) interpretation: the expression change of experimental group and matched group withRepresent.
5. result
(1) crowd circulates the expression of miRNAs
The circulation miRNAs that table 1 obesity patient's expression changes
Note: in table, numerical value represents with the fluorescence intensity after quantile method normalized.
Multiple changes=[(2Fat group 1+2Fat group 2)/2]/[(2Regular restructuring 1+2Regular restructuring 2)/2]。
For getting rid of individual variation as far as possible to the impact of experimental result, the miRNAs that is enriched with most differential expression, by fertilizer
Fat disease patient's group, normal type matched group respectively make 2 random biased samples.Fold difference (fold changes, FC) > 3 or FC
< miRNAs of-3 includes analysis further in.Chip results shows, has 34 miRNAs tables in simple obesity patients serum
The amount of reaching generation significance changes (table 1)；Wherein, have 4 miRNAs to express under notable rise, 30 miRNAs expression significantly
Adjust.In the miRNAs that all expression significances change, Microrna-122 (microRNA-122, miR-122) is circulation
The miRNA that middle gene expression abundance is the highest；Previously research shows, under liver metabolism disturbance state, miR-122 expression is lacked of proper care；Its
Regulation plasma cholesterol and lipids contents in Mice Body.Therefore, circulation miR-122 expression is selected to support with fat and insulin
Anti-relation.
(2) obesity patient circulates the rise of miR-122 expression
Quantitative fluorescent PCR is consistent with micro-array chip result, than normal type matched group, verifies crowd obesity patient
Serum miR-122 level is increased to 3.07 ± 0.24 times of (P=7.10 × 10-12, Figure 1A).Obesity patient organizes serum miR-122
CT value be 30.85 ± 1.17, regular restructuring serum miR-122 CT value be 32.37 ± 1.55 (Figure 1B).Correction sex,
After age, blood fat and liver function, obesity patient organize circulation miR-122 level be still significantly higher than normal type matched group (P <
0.001, Fig. 1 C).
For probing into circulation miR-122 level and the fat relation occurred further, obesity patient is divided into Mild Obesity
Group, central obesity group and severe simple obesity group.Compared with normal type matched group, Mild Obesity group, central obesity group and severe are fertile
Fat group of circulation miR-122 level significantly raises (P equal < 0.05, Fig. 2 A)；Circulation miR-122 level presents and adds with obese degree
The trend weighed and raise, although not having significant difference.After correction sex, age, blood fat and liver function, than normal type
Matched group, Mild Obesity group, central obesity group, severe simple obesity group circulation miR-122 level significantly raises (P < 0.01, figure
2B).(3) circulation miR-122 level and the correlation analysis of multiple variablees
Table 2 circulates miR-122 level and the dependency of each variable and stepwise regression analysis showed
Note: r, Pearson correlation coefficient；β, standard regression coefficient；"-" represents that this variable is introduced into optimal regression equation.
Pearson Correlation analysis showed, circulation miR-122 level and BMI, waist-to-hipratio, body fat content, blood pressure, liver enzyme,
TG, HDL-c, fasting glucose and insulin, OGTT-2h blood glucose and insulin, HOMA-IR, sex significant correlation (P all < 0.01,
Table 2).Stepwise regression analysis showed finds, circulation miR-122 level and Serum ALT levels independence the most relevant (β=0.648, P <
0.001；Table 2).
Table 3 normal type experimenter circulates miR-122 level and the dependency of each variable and stepwise regression analysis showed
Note: r, Pearson correlation coefficient；β, standard regression coefficient；"-" represents that this variable is introduced into optimal regression equation.
In view of regular restructuring exists at aspects such as insulin sensitivity, blood glucose regulation, liver functions with obesity patient's group
More significantly difference, we analyze circulation miR-122 level respectively with each in regular restructuring and obesity patient's group
The dependency of metabolic index.In normal type experimenter, circulation miR-122 expression and ALT, ALP, γ-GT, HDL-c,
Blood glucose, fasting insulin, sex significant correlation (P all < 0.05, table 3).Stepwise regression analysis showed shows, circulates miR-122 table
The level that reaches independent with sex (β=-0.425, P < 0.001) and HDL-c (β=-0.287, P < 0.001) relevant (table 3).
Table 4 obesity patient circulates miR-122 level and the dependency of each variable and stepwise regression analysis showed
Note: r, Pearson correlation coefficient；β, standard regression coefficient；"-" represents that this variable is introduced into optimal regression equation.
In obesity patient, circulation miR-122 expression and ALT, AST, γ-GT, TG and blood glucose significant correlation (P
All < 0.05, table 4).Multivariate regressive analysis shows, circulation miR-122 expression and Serum ALT levels independence phase
Close (β=0.554, P < 0.001；Table 4).
Result shows, people circulates miR-122 level and liver enzyme level significant correlation.Additionally, stepwise regression analysis showed is sent out
Existing, it is independent to serum ALT concentration relevant that people circulates miR-122 level.Wang etc. study discovery, mice plasma miR-122 concentration
Dose dependent, open-assembly time dependency is become relevant to ALT level, hepatopathy；Than ALT level, circulate miR-122 level
The sensitivity, the credibility that change are higher.Later research can compare human circulation miR-122 level, the spirit of ALT level further
Sensitivity, credibility, thus promote the clinical practice of circulation miR-122 level.Additionally, the basic research in future can be studied further
Whether circulation miR-122 has the biological function of regulation and control insulin sensitivity.
Claims (5)
1. people circulates a detection method of miR-122, including:
Sample, as sample, is dissolved in the distilled water of nuclease, takes Partial Inverse and be transcribed into cDNA, enter by extraction serum total serum IgE
Row miR-122 specificity real-time fluorescence quantitative PCR reacts, and obtains the result that chip reflects by Ct value and verifies,.
A kind of people the most according to claim 1 circulates the detection method of miR-122, it is characterised in that: described reverse transcription is adopted
The consisting of of reverse transcription mixed liquor: 5 × reverse transcription reaction liquid 2 μ l, reverse transcriptase mix 1 μ l, Quality Control Spike-In is molten
Liquid 0.5 μ l, removes the distilled water 4.5 μ l of nuclease.
A kind of people the most according to claim 1 circulates the detection method of miR-122, it is characterised in that: described reverse transcription bar
Part is: 42 DEG C of reaction 1h, and 95 DEG C of reaction 5min are subsequently reduced to 4 DEG C.
A kind of people the most according to claim 1 circulates the detection method of miR-122, it is characterised in that: described real-time fluorescence
The reaction system of quantitative PCR is: cDNA template 4 μ l, primer 1 μ l, 2 × SYBR 5 μ l.
A kind of people the most according to claim 1 circulates the detection method of miR-122, it is characterised in that: described real-time fluorescence
The reaction condition of quantitative PCR is: 95 DEG C of denaturations 10min；95 DEG C of 10s, 60 DEG C extend 1min, 45 circulations.
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Application publication date: 20161026 |