CN101368213A - Blood serum minuteness ribonucleic acid reagent kit and application in early diagnosis of hepatitis B - Google Patents
Blood serum minuteness ribonucleic acid reagent kit and application in early diagnosis of hepatitis B Download PDFInfo
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Abstract
invention discloses a kit for a serum micro-ribonucleic acid and the application in the early diagnosing of hepatitis B thereof which belong to the technical field of biotechnological pharmaceutics. The invention provides the combination of the micro-ribonucleic acid used for estimating the physiological and/or pathological states of a normal person, a HBVer and a patient with hepatitis B; the combination comprises all the micro-ribonucleic acids which have differential expression in the serum/plasm of the normal person, the HBVer and the patient with hepatitis B and prepares a diagnosing kit which can be used for the early diagnosing of hepatitis B. The combination and the method can be used for the early detection and nondirective therapy of hepatitis B; besides, the combination and the method have the advantages of high specificity, high efficiency, high sensitivity, low detection cost, convenient material obtaining, and the like. Besides, the samples are easily stored.
Description
One technical field
The invention belongs to the biotechnological pharmaceutics technical field, be specifically related to of the application of blood serum miRNA at aspects such as the screening of the diagnosis of hepatitis B, prediction, therapeutic evaluation, active constituents of medicine, evaluating drug effects.
Two background technologies
Hepatitis B virus (Hepatitis B virus:HBV) is one of modal pathogenic microorganism in the whole world.According to statistics, the whole world has 400,000,000 hepatitis B viruses (HBV) carrier approximately at present.Chronic hepatitis B may cause severe complications (as liver cirrhosis and liver cancer), and statistic data shows that the primary hepatocarcinoma in the whole world about 80% is with hepatitis b virus infected relevant.Epidemiology survey shows that China has 1.3 hundred million people to carry hepatitis B virus for a long time approximately.The expert calculates, the existing chronic viral hepatitis B patient of China is about more than 2,000 ten thousand, and annual have 280,000 people to die from liver cirrhosis relevant with hepatitis B virus infection or liver cancer approximately.At present traditional biological chemistry and the Protocols in Molecular Biology method that hepatitis B is carried out clinical diagnosis mainly comprises reverse passive hemagglutination, enzyme linked immunosorbent assay, gene diagnosis etc., these methods are also more loaded down with trivial details and coarse, all can have various defectives or deficiency aspect sensitivity, specificity, efficiency, popularization, sample preparation and the preservation.Because hepatitis B virus infection does not still have the ideal method of early diagnosis, therefore developing effective hepatitis B detection method is the task of top priority, could realize diagnosis morning, the early treatment of hepatitis B patient like this.
MiRNA, English microRNA by name is the non-coding strand micro ribonucleic acid molecule that a class is about 19 to 23 Nucleotide.They are high conservative on evolving, and with many normal physiological activity of animal, closely related as biont growth, tissue differentiation, natural death of cerebral cells and energy metabolism etc., also exist closely simultaneously and get in touch with the generation of numerous disease and development.The nearest expression level of discovering several miRNAs in lymphocytic leukemia and the Burkitt lymphoma all has downward modulation in various degree; When analyzing the miRNA expression of comparing in people's lung cancer, the breast cancer tissue, finding has the expression level of some tissue specificity miRNAs with respect to healthy tissues variation to take place.There are some researches prove that also miRNA has influenced the generation and the development of cardiovascular disordeies such as myocardial hypertrophy, heart failure, atherosclerosis, and close association is arranged with metabolic diseases such as type ii diabetes.Exist positive connection between these experimental result prompting miRNA expression and specific variations and disease generation and the development.
Owing to play vital role beyond imagination in the expression regulation of miRNA after genetic transcription, therefore the cognation below it exists with disease: at first, the variation of miRNA may be the cause of disease, this is because the supressor of disease and the promotion factor all may be the target sites of miRNA, when disorderly expression has taken place earlier miRNA itself, promote the miRNA expression amount of the factor to reduce such as the original disease that suppresses, the miRNA expression amount that perhaps suppresses the disease supressor has raise, its net result all can cause the whole disorderly of downstream series of genes change of Expression and some path, and then the generation that induces an illness; Secondly, the variation of miRNA also may be the result of disease, this is because when disease (as cancer) takes place, can cause the losing of chromosome segment, the sudden change of gene or the violent amplification of chromosome segment, if miRNA just in time is positioned at this varied sections, its expression amount will take place to change extremely significantly so.Therefore, the miRNA molecule can be used as the new disease marker of a class fully in theory, and its specific variations is inevitable to be associated with disease generation development.MiRNA can also by miRNA that suppresses to raise in the lysis and the miRNA of crossing down-regulated expression, might greatly be alleviated the generation and the development of disease as the potential drug target simultaneously.
The inventor has carried out with the research of miRNA as the association area of disease marker, and for example we find that most blood serum minuteness ribonucleic acid comes from the cell particles (microparticles) in the serum.Cytolemma micropartical in the serum is from the different cell of human body (being not only hemocyte) and comprise the miRNA relevant with specific cells/tissue, and the variation that detects the interior miRNA of cytolemma micropartical in the serum just can obtain the pathology situation of whole machine body; The more important thing is that cell particles has distinctive receptor protein of source cell surface or film fat structure, with corresponding target cell high-affinity is arranged, thereby can optionally send miRNA, thereby the effect that improves miRNA regulating cell function greatly to target cell/tissue.Obviously, because cell particles (or similar atomic film lipid vesicle structure) itself has and particular organization and cell bonded specificity, its contained miRNA also presents stronger target, stability and high-level efficiency, and it is having the major application prospect aspect the diagnosis of disease and the treatment.We also choose hepatitis B as research object in addition, find after deliberation, between normal people, hepatitis B carriers, hepatitis B patient, some miRNA molecules all exist specific variations, and have set up a kind of more responsive, more accurate method of making a definite diagnosis, treating hepatitis B in early days by the specific variations of measuring miRNA in view of the above.
Three summary of the invention
At present, the diagnosis of hepatitis B only is confined to various biochemical indicators in the blood serum, also is not used for any report of diagnosis of hepatitis b and treatment aspect about the blood serum miRNA.Based on this discovery of miRNA, the inventor will study sight and be locked in the major application prospect of blood serum miRNA aspect diagnosis of hepatitis b and treatment.
The inventor seeks the high efficiency miRNA of target that a class can be used for treating hepatitis B by the miRNA in separation and research normal people, HBVer and the hepatitis B patient serum.We have adopted two kinds of research techniques: the one, get the some normal peoples of 40ml, HBVer, hepatitis B patient's serum respectively, and extract total RNA and carry out the Solexa order-checking.The 2nd, the serum with each normal people, HBVer and hepatitis B patient removes albumen respectively, and supernatant carries out quantitative PCR detection.Because the miRNA molecule is the novel disease marker of a class, therefore wish by whether the blood serum miRNA is existed, difference between the individuality and with the research of the dependency of hepatitis B, set up a kind of miRNA of stable existence in the blood serum and specific variations thereof utilized and carry out the early diagnosis of hepatitis B, course of disease monitoring, the prediction that complication takes place, drug effect is judged, medicine guide, individualized treatment, the effective components of Chinese medicinal screening, the new technology of researchs such as kind heap sort.
The problem that the present invention need solve comprises: miRNA molecular distribution and specificity thereof in (1) Analysis and Identification normal people, HBVer, the hepatitis B patient blood serum; (2) specific variations of blood serum miRNA in the pathogenic process of research hepatitis B; The class blood serum miRNA molecule that the differential expression degree under hepatitis B patient, HBVer and normal physiological state that (3) will screen is big is used for the development of blood serum miRNA detection technique, prepares the biochip or the diagnostic kit that are applied to hepatitis diagnosis.(4) external specific miRNA is delivered to specific cells/tissue, function of organization improves or the effect of disease treatment thereby reach.
The invention provides and a kind ofly have higher target, susceptibility, stability and high-level efficiency and at the miRNA that has the major application prospect aspect the diagnosis of hepatitis B and the treatment.The technical scheme of its research mainly comprises following components:
1.Solexa order-checking
(1) collects the blood serum sample: collect normal people, HBVer, the some parts of hepatitis B patient serum, from every portion of normal human serum, take out the extraction that a part of pooled serum that blendes together 40ml is used for total RNA.HBVer and hepatitis B patient also are like this.
(2) extract the total RNA of blood serum by Trizol reagent:
The every 1ml serum of a adds the Trizol (solexa need about 40ml serum) of 1ml, shakes mixing with hand;
B is separated: hatched homogenate 5 minutes for 15-30 ℃, cause the nucleoprotein complex body fully to dissociate, the 1ml/200ul chloroform covers tight lid, firmly shakes 15 seconds, hatched 2-3 minute for 15-30 ℃, (132,000rpm) centrifugal 15 minutes, centrifugal back mixture was divided into three layers: lower floor's phenol chloroform phase, intermediate phase, colourless upper strata water-contain RNA for 2-8 ℃ of (4 ℃) 10000rcf, RNA is dissolved in the upper strata water fully, and the upper strata water accounts for 60% of cumulative volume;
Unnecessary albumen is removed in the extracting of c multistep phenol-chloroform: 1/3 volume phenol (removing DNA albumen) concussion, and centrifugal 5 minutes of 10000g shifts supernatant; 1/6 volume phenol+1/6 volume chloroform concussion, centrifugal 5 minutes of 10000g shifts supernatant; The concussion of 1/3 volume chloroform, centrifugal 5 minutes of 10000g shifts supernatant, removes unnecessary phenol;
D RNA precipitation: with the upper water phase transition, amount by every 1ml Trizol/500ul Virahol adds Virahol (general excessive Virahol, can be added to the containment of centrifuge tube), ice bath or 4 ℃ were placed 1 hour, centrifugal 1 hour of 2-8 ℃ of (4 ℃) 10000rcf, then the RNA crude extract will be deposited at the bottom of the official, and Virahol is sopped up with rifle;
Attention: that Virahol adds is excessive (as seen 25ml+15ml is deposited under the light, film like)
E leaves standstill for a moment up to resolution of precipitate on ice with Trizol dissolution precipitation (can more clearly see precipitation this moment, the is the dewdrop shape) piping and druming of 1ml, is transferred in the EP pipe;
F repeats the 2-3 step, if albumen impurity is few, can dispense step 3;
G RNA precipitation: with the upper water phase transition, amount by every 1ml Trizol/500ul Virahol adds Virahol, 15-30 ℃ of hatching (ice bath) 10 minutes, 2-8 ℃ of (4 ℃) 10000rcf (132,000rpm) centrifugal 10 minutes then the RNA crude extract will be deposited in the pipe end;
H washes RNA: outwell supernatant, the alcohol flushing with 75% precipitates once, and consumption is a 1mlTrizol/1ml ethanol, with the resuspended milky white precipitate of oscillator, precipitation comes off at the end floating from pipe, 2-8 ℃ of (4 ℃) 10000rcf (132,000rpm) centrifugal 5 minutes;
I dissolves RNA: sop up supernatant with rifle, and slightly once centrifugal, supernatant is thoroughly blotted only, dry 5 minutes (transparent or semitransparent state), be sure not to make the RNA complete drying, otherwise RNA is difficult to dissolving.Dissolving back (about 20ul) is in-70 ℃ of preservations;
J measures concentration.
(3) carry out the PAGE electrophoresis and reclaim 17-27nt RNA molecule;
(4) adaptor prime enzyme being associated in 3 ' and 5 ' of small RNA molecular holds;
(5) carry out RT-PCR reaction back and checking order;
(6) data analysis and processing.
According to the detected normal people who there are differences expression of aforesaid method, the HBVer, miRNA in the hepatitis B patient blood serum comprises let-7c, miR-1, miR-10a, miR-122, miR-125b, miR-128a, miR-128b, miR-150, miR-197, miR-221, miR-222, miR-223, miR-23a, miR-23b, miR-27a, miR-27b, miR-30a, miR-342-3p, miR-361-5p, miR-423-5p, miR-532-5p, miR-574-3p, miR-629, miR-92a, miR-92b, miR-99a, miR-139-5p, miR-193a-5p, miR-193b, miR-365, miR-375, miR-455-3p, miR-483-3p, miR-483-5p, miR-486-3p, miR-885-5p, miR-99b, let-7f, let-7g, let-7i, miR-101, miR-103, miR-106a, miR-106b, miR-107, miR-126, miR-130a, miR-130b, miR-142-3p, miR-142-5p, miR-144, miR-146a, miR-146b-5p, miR-148b, miR-151-5p, miR-15a, miR-16, miR-17, miR-182, miR-183, miR-185, miR-186, miR-18a, miR-191, miR-19b, miR-20a, miR-20b, miR-21, miR-210, miR-26a, miR-26b, miR-29c, miR-30e, miR-340, miR-362-5p, miR-363, miR-374a, miR-378, miR-424, miR-451, miR-454, miR-532-5p, miR-652, miR-660, miR-7, miR-923, miR-93, miR-96, miR-98.
2.Real-time PCR method
(1) extraction experimenter's the total RNA of blood serum obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
(2) design primer with miRNA;
(3) add fluorescent probe and carry out the PCR reaction;
(4) variation of the amount of miRNA in detection and comparison normal people, HBVer, the hepatitis B patient blood serum sample.
We have selected to have between normal people and HBVer and the hepatitis B patient let-7c than big-difference as a result according to solexa, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, let-7f, miR-423-5p, miR-574-3p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p, miR-885-5p, miR-101, miR-106b, miR-16, miR-185, miR-451 comprises let-7c with the miRNA that there are differences expression in the detected HBVer of Real-time PCR method and the normal human serum/blood plasma, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p, miR-885-5p, the miRNA that there are differences expression in hepatitis B patient and the normal human serum/blood plasma comprises let-7c, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p, miR-885-5p, the miRNA that there are differences expression in HBVer and the hepatitis B patient blood serum comprises let-7c, miR-423-5p, miR-139-5p.
3. the present invention provides also that a kind of to be used to estimate the experimenter be the normal people, the HBVer still is the test kit of hepatitis B patient, described test kit can comprise the normal people, the HBVer, there are differences the primer of the whole ripe body miRNA of expression in the hepatitis B patient blood serum, specifically comprise let-7c, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, the primer of miR-483-5p and miR-885-5p.
Particularly, in aforesaid combination, method, the test kit, be used to diagnose and/or differentiate the experimenter physiology and/or pathological state, be used to screen determinand the activity that prevents and/or treats hepatitis B, estimate validity that the experimenter is treated, complication is taken place the experimenter predict.
At present traditional biological chemistry and the Protocols in Molecular Biology method that hepatitis B is carried out clinical diagnosis mainly comprises reverse passive hemagglutination, enzyme linked immunosorbent assay, gene diagnosis etc., these methods are also more loaded down with trivial details and coarse, all can have various defectives or deficiency aspect sensitivity, specificity, efficiency, popularization, sample preparation and the preservation.That traditional treating hepatitis B method mainly comprises is antiviral, immunomodulatory, anti-inflammatory protect the liver, anti-fibrosis and symptomatic treatment etc., wherein antiviral therapy is crucial, mainly comprises interferon therapy, nucleoside analog treatment, immune modulating treatment and other antiviral and treatment by Chinese herbs.But because hepatitis B virus has the individual specificity, all there are certain side effect in these traditional methods of treatment, cause the resistance of body and curative effect all lower.Blood serum miRNA detection technique, biochip and diagnostic kit based on the blood serum miRNA are combined as a whole the peculiar property and the conventional molecular Biological Detection technology of blood serum miRNA dexterously, they analyze the composition of miRNA in HBVer or the patients serum/blood plasma in high-throughput ground apace, and clinical applicability is extremely strong.Because the physiological status variation of organ-tissue can cause the change that the blood serum miRNA is formed, so the blood serum miRNA can be used as " disease fingerprint ", is used for the early diagnosis of hepatitis B.
The superiority of blood serum miRNA detection technique:
(1) the blood serum miRNA is as novel disease marker, have the pedigree of detecting wide, highly sensitive, detect cost low, draw materials conveniently, sample advantages such as (blood serum-20 ℃ deposit get final product) easy to store, this method can be widely used in related works such as hepatitis B generaI investigation, becomes the effective means of early diagnosis hepatitis B.
(2) the blood serum miRNA will improve low specificity and the muting sensitivity that individual difference that single marker is difficult to overcome is brought as new disease markers, significantly improve the clinical recall rate of hepatitis B and realize the early stage diagnosis and treatment of hepatitis B.
What (3) advantage of blood serum miRNA detection technique was its detection is a series of disease-related marker, thereby can overcome difference (being age, sex, race, diet and environment etc.) between the individual patient, and this single just disease markers a subject matter can't going beyond.
Four embodiments
The Solexa of miRNA order-checking in embodiment 1 normal people, HBVer, the hepatitis B patient blood serum, concrete steps are:
(1) collects the blood serum sample: collect normal people, HBVer, the some parts of hepatitis B patient serum, from every portion of normal human serum, take out the extraction that a part of pooled serum that blendes together 40ml is used for total RNA.HBVer and hepatitis B patient also are like this.
(2) extract the total RNA of blood serum by Trizol reagent:
The every 1ml serum of a adds the Trizol (solexa need about 40ml serum) of 1ml, shakes mixing with hand;
B is separated: hatched homogenate 5 minutes for 15-30 ℃, cause the nucleoprotein complex body fully to dissociate, the 1ml/200ul chloroform covers tight lid, firmly shakes 15 seconds, hatched 2-3 minute for 15-30 ℃, (132,000rpm) centrifugal 15 minutes, centrifugal back mixture was divided into three layers: lower floor's phenol chloroform phase, intermediate phase, colourless upper strata water-contain RNA for 2-8 ℃ of (4 ℃) 10000rcf, RNA is dissolved in the upper strata water fully, and the upper strata water accounts for 60% of cumulative volume;
Unnecessary albumen is removed in the extracting of c multistep phenol-chloroform: 1/3 volume phenol (removing DNA albumen) concussion, and centrifugal 5 minutes of 10000g shifts supernatant; 1/6 volume phenol+1/6 volume chloroform concussion, centrifugal 5 minutes of 10000g shifts supernatant; The concussion of 1/3 volume chloroform, centrifugal 5 minutes of 10000g shifts supernatant, removes unnecessary phenol;
D RNA precipitation: with the upper water phase transition, amount by every 1ml Trizol/500ul Virahol adds Virahol (general excessive Virahol, can be added to the containment of centrifuge tube), ice bath or 4 ℃ were placed 1 hour, centrifugal 1 hour of 2-8 ℃ of (4 ℃) 10000rcf, then the RNA crude extract will be deposited at the bottom of the official, and Virahol is sopped up with rifle;
Attention: that Virahol adds is excessive (as seen 25ml+15ml is deposited under the light, film like)
E leaves standstill for a moment up to resolution of precipitate on ice with Trizol dissolution precipitation (can more clearly see precipitation this moment, the is the dewdrop shape) piping and druming of 1ml, is transferred in the EP pipe;
F repeats the 2-3 step, if albumen impurity is few, can dispense step 3;
G RNA precipitation: with the upper water phase transition, amount by every 1ml Trizol/500ul Virahol adds Virahol, 15-30 ℃ of hatching (ice bath) 10 minutes, 2-8 ℃ of (4 ℃) 10000rcf (132,000rpm) centrifugal 10 minutes then the RNA crude extract will be deposited in the pipe end;
H washes RNA: outwell supernatant, the alcohol flushing with 75% precipitates once, and consumption is a 1mlTrizol/1ml ethanol, with the resuspended milky white precipitate of oscillator, precipitation comes off at the end floating from pipe, 2-8 ℃ of (4 ℃) 10000rcf (132,000rpm) centrifugal 5 minutes;
I dissolves RNA: sop up supernatant with rifle, and slightly once centrifugal, supernatant is thoroughly blotted only, dry 5 minutes (transparent or semitransparent state), be sure not to make the RNA complete drying, otherwise RNA is difficult to dissolving.Dissolving back (about 20ul) is in-70 ℃ of preservations;
J measures concentration.
(3) carry out the PAGE electrophoresis and reclaim 17-27nt RNA molecule;
(4) adaptor prime enzyme being associated in 3 ' and 5 ' of small RNA molecular holds;
(5) carry out RT-PCR reaction back and checking order;
(6) data analysis and processing
Concrete experimental result sees the following form the relative copy number of numeric representation microRNA wherein.
| microRNA | normal | HBV | HBV-positive |
| hsa-let-7c | 13.15945 | 1073.093 | 675.4264 |
| hsa-miR-1 | 2.732031 | 20.46152 | 2.837926 |
| hsa-miR-10a | 2.674524 | 52.29055 | 5.675852 |
| hsa-miR-122 | 2.838615 | 69500.97 | 27195.85 |
| hsa-miR-125b | 2.988329 | 88.66659 | 122.0308 |
| hsa-miR-128a | 8.317054 | 25.00853 | 70.94815 |
| hsa-miR-128b | 8.317054 | 20.46152 | 62.43437 |
| hsa-miR-150 | 8.987792 | 120.4956 | 122.0308 |
| hsa-miR-197 | 1.462437 | 9.094009 | 48.24474 |
| hsa-miR-221 | 6.912123 | 88.66659 | 167.4376 |
| hsa-miR-222 | 14.29619 | 56.83756 | 122.0308 |
| hsa-miR-223 | 41.02111 | 168.2392 | 493.7991 |
| hsa-miR-23a | 12.94629 | 529.726 | 743.5366 |
| hsa-miR-23b | 5.820996 | 202.3417 | 244.0616 |
| hsa-miR-27a | 2.432603 | 34.10253 | 56.75852 |
| hsa-miR-27b | 8.08108 | 40.92304 | 36.89304 |
| hsa-miR-30a | 2.40385 | 9.094009 | 25.54133 |
| hsa-miR-342-3p | 6.975577 | 152.3247 | 139.0584 |
| hsa-miR-361-5p | 0.328181 | 0 | 28.37926 |
| hsa-miR-423-5p | 198.008 | 5740.593 | 6297.358 |
| hsa-miR-532-3p | 3.35964 | 2.273502 | 25.54133 |
| hsa-miR-574-3p | 0.656362 | 9.094009 | 110.6791 |
| hsa-miR-629 | 2.910498 | 59.11106 | 19.86548 |
| hsa-miR-92a | 339.4713 | 3355.689 | 6572.637 |
| hsa-miR-92b | 2.090046 | 15.91452 | 42.56889 |
| hsa-miR-99a | 1.255217 | 127.3161 | 198.6548 |
| hsa-miR-139-5p | 0 | 22.73502 | 107.8412 |
| hsa-miR-193a-5p | 0 | 2.273502 | 36.89304 |
| hsa-miR-193b | 0 | 43.19654 | 34.05511 |
| hsa-miR-365 | 0 | 6.820507 | 51.08267 |
| hsa-miR-375 | 0 | 147.7777 | 184.1652 |
| hsa-miR-455-3p | 0 | 11.36751 | 62.43437 |
| hsa-miR-483-3p | 0 | 25.00853 | 8.513778 |
| hsa-miR-483-5p | 0 | 25.00853 | 130.5446 |
| hsa-miR-486-3p | 0 | 13.64101 | 31.21719 |
| hsa-miR-885-5p | 0 | 47.74355 | 201.4927 |
| hsa-miR-99b | 0 | 47.74355 | 45.40682 |
| hsa-let-7f | 2009.114 | 250.0853 | 229.872 |
| hsa-let-7g | 2058.343 | 204.6152 | 119.1929 |
| hsa-let-7i | 1075.084 | 86.39309 | 82.29986 |
| hsa-miR-101 | 2510.382 | 9.094009 | 14.18963 |
| hsa-miR-103 | 1002.754 | 90.94099 | 822.9986 |
| hsa-miR-106a | 157.5739 | 4.547005 | 79.46193 |
| hsa-miR-106b | 1400.943 | 2.273502 | 31.21719 |
| hsa-miR-107 | 544.5387 | 34.10253 | 471.0957 |
| hsa-miR-126 | 34.48625 | 0 | 2.837926 |
| hsa-miR-130a | 116.1274 | 2.273502 | 11.3517 |
| hsa-miR-130b | 59.45773 | 2.273502 | 19.86548 |
| hsa-miR-142-3p | 326.8225 | 2.273502 | 8.513778 |
| hsa-miR-142-5p | 548.7694 | 13.64101 | 31.21719 |
| hsa-miR-144 | 317.7534 | 0 | 25.54133 |
| hsa-miR-146a | 108.8009 | 18.18802 | 28.37926 |
| hsa-miR-146b-5p | 45.10652 | 2.273502 | 22.70341 |
| hsa-miR-148b | 32.77594 | 4.547005 | 19.86548 |
| hsa-miR-151-5p | 200.3241 | 15.91452 | 68.11023 |
| hsa-miR-15a | 465.229 | 38.64954 | 113.517 |
| hsa-miR-16 | 6805.839 | 377.4014 | 1231.66 |
| hsa-miR-17 | 556.7146 | 20.46152 | 297.9822 |
| hsa-miR-182 | 77.76993 | 0 | 0 |
| hsa-miR-183 | 36.89853 | 0 | 0 |
| hsa-miR-185 | 1848.542 | 61.38456 | 184.4652 |
| hsa-miR-186 | 338.3713 | 2.273502 | 11.3517 |
| hsa-miR-18a | 119.6943 | 0 | 0 |
| hsa-miR-191 | 637.0119 | 90.94009 | 360.4166 |
| hsa-miR-19b | 483.5556 | 0 | 14.18963 |
| hsa-miR-20a | 1484.046 | 0 | 34.05511 |
| hsa-miR-20b | 98.66692 | 2.273502 | 11.3517 |
| hsa-miR-21 | 933.8693 | 102.3076 | 133.3825 |
| hsa-miR-210 | 30.34929 | 0 | 2.837926 |
| hsa-miR-26a | 550.3696 | 36.37604 | 158.9239 |
| hsa-miR-26b | 284.1178 | 15.91452 | 56.75852 |
| hsa-miR-29c | 105.0243 | 2.273502 | 2.837926 |
| hsa-miR-30e | 79.16643 | 4.547005 | 8.513778 |
| hsa-miR-340 | 108.954 | 0 | 2.837926 |
| hsa-miR-362-5p | 26.31296 | 0 | 2.837926 |
| hsa-miR-363 | 310.6821 | 2.273502 | 45.40682 |
| hsa-miR-374a | 39.61618 | 2.273502 | 2.837926 |
| hsa-miR-378 | 170.4324 | 61.38456 | 17.02756 |
| hsa-miR-424 | 22.87549 | 2.273502 | 0 |
| hsa-miR-451 | 58299.32 | 2193.93 | 25050.37 |
| hsa-miR-454 | 22.03472 | 0 | 0 |
| hsa-miR-532-5p | 31.37696 | 6.820507 | 2.837926 |
| hsa-miR-652 | 45.45156 | 4.547005 | 45.40682 |
| hsa-miR-660 | 28.65336 | 2.273502 | 17.02756 |
| hsa-miR-7 | 476.6444 | 4.547005 | 0 |
| hsa-miR-923 | 28.86999 | 6.820507 | 2.837926 |
| hsa-miR-93 | 510.0307 | 70.47857 | 85.13778 |
| hsa-miR-96 | 24.10444 | 0 | 0 |
| hsa-miR-98 | 65.4225 | 0 | 2.837926 |
Part there are differences the real-time PCR experiment of the miRNA of expression in embodiment 2 normal peoples, HBVer, the hepatitis B patient blood serum
The experimental procedure of quantitative PCR is as follows:
(1) extraction experimenter's the total RNA of blood serum obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
(2) design primer with miRNA;
(3) add fluorescent probe and carry out the PCR reaction;
(4) variation of the amount of miRNA in detection and comparison normal people, HBVer, the hepatitis B patient blood serum sample.
The dyestuff that quantitative PCR experiment is used is EvaGreen, and what instrument used is ABI Prism 7500 quantitative real time PCR Instruments, and reaction conditions is to carry out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carries out 40 circulations in 60 ℃, 1 minute.Normal people, carrier, hepatitis B patients serum/plasma sample and normal human serum/plasma sample are directly carried out reverse transcription reaction with phenol and chloroform except that behind the albumen, react the wherein amount of contained miRNA by quantitative PCR.We according to solexa result choose let-7c, miR-10a, miR-122, niR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, let-7f, miR-423-5p, miR-574-3p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p, miR-885-5p, miR-101, miR-106b, miR-16, miR-185, miR-451 experimentize, experimental result sees the following form 1,2:
Table 1 carrier and normal people's comparison
| microRNA | Normal people's mean value | SD | Carrier's mean value | TTEST (comparing) with the normal people | SD |
| let-7c | 1 | 0.07 | 4.50 | 6.87E-21 | 0.34 |
| 10a | 1 | 0.08 | 134.80 | 6.35E-34 | 8.35 |
| 122 | 1 | 0.13 | 94.24 | 9.83E-19 | 8.49 |
| 125b | 1 | 0.11 | 12.42 | 1.96E-08 | 1.79 |
| 150 | 1 | 0.12 | 7.45 | 1.27E-06 | 1.20 |
| 223 | 1 | 0.07 | 4.60 | 2.74E-40 | 0.20 |
| 23a | 1 | 0.09 | 15.81 | 1.39E-12 | 1.82 |
| 23b | 1 | 0.12 | 16.40 | 1.75E-09 | 2.35 |
| 27a | 1 | 0.16 | 48.41 | 3.00E-19 | 4.33 |
| 342-3p | 1 | 0.06 | 25.77 | 2.98E-20 | 2.31 |
| Let-7f | 1 | 0.09 | 346.82 | 1.56E-36 | 19.86 |
| 423-5p | 1 | 0.07 | 39.01 | 4.03E-06 | 8.00 |
| 574-3p | 1 | 0.06 | 1.50 | 0.000552 | 0.13 |
| 92a | 1 | 0.09 | 2.65 | 1.14E-08 | 0.27 |
| 99a | 1 | 0.12 | 11.95 | 6.11E-20 | 1.05 |
| 139-5p | 1 | 0.08 | 1.71 | 0.000233 | 0.17 |
| 375 | 1 | 0.06 | 106.78 | 2.33E-18 | 11.07 |
| 483-5p | 1 | 0.10 | 222.70 | 1.13E-28 | 15.93 |
| 885-5p | 1 | 0.05 | 5.00 | 2.33E-16 | 0.44 |
| 101 | 1 | 0.13 | 31.05 | 6.88E-35 | 1.92 |
| 106b | 1 | 0.13 | 69.42 | 3.31E-08 | 11.18 |
| 16 | 1 | 0.12 | 40.02 | 1.57E-13 | 4.81 |
| 185 | 1 | 0.06 | 20.19 | 3.13E-30 | 1.43 |
| 451 | 1 | 0.10 | 7.49 | 3.39E-20 | 0.61 |
Table 2 patient and normal people and carrier's comparison
| microRNA | Normal people's mean value | SD | Patient's mean value | TTEST (comparing) with the normal people | TTEST (compares with the carrier | SD |
| let-7c | 1 | 0.07 | 3.18 | 4.35E-19 | 0.002108 | 0.23 |
| 10a | 1 | 0.08 | 136.93 | 3.79E-33 | 0.865456 | 9.11 |
| 122 | 1 | 0.13 | 104.96 | 9.83E-25 | 0.370079 | 8.19 |
| 125b | 1 | 0.11 | 12.61 | 7.52E-23 | 0.928597 | 0.98 |
| 150 | 1 | 0.12 | 12.09 | 2.05E-09 | 0.028423 | 1.73 |
| 223 | 1 | 0.07 | 4.45 | 1.20E-51 | 0.559124 | 0.15 |
| 23a | 1 | 0.09 | 18.14 | 8.07E-06 | 0.564329 | 3.70 |
| 23b | 1 | 0.12 | 14.91 | 2.07E-15 | 0.607368 | 1.62 |
| 27a | 1 | 0.16 | 60.60 | 5.78E-10 | 0.207239 | 8.88 |
| 342-3p | 1 | 0.06 | 29.69 | 4.52E-33 | 0.205942 | 1.96 |
| Let-7f | 1 | 0.09 | 186.99 | 1.60E-15 | 1.76E-06 | 21.03 |
| 423-5p | 1 | 0.07 | 18.72 | 6.34E-09 | 0.023956 | 3.07 |
| 574-3p | 1 | 0.06 | 1.62 | 1.76E-05 | 0.518277 | 0.13 |
| 92a | 1 | 0.09 | 2.54 | 2.10E-16 | 0.724386 | 0.15 |
| 99a | 1 | 0.12 | 10.94 | 6.04E-40 | 0.414554 | 0.58 |
| 139-5p | 1 | 0.08 | 1.23 | 0.027781 | 0.010982 | 0.07 |
| 375 | 1 | 0.06 | 94.62 | 1.56E-34 | 0.356989 | 6.56 |
| 483-5p | 1 | 0.10 | 136.68 | 1.57E-20 | 0.000152 | 12.84 |
| 885-5p | 1 | 0.05 | 5.59 | 2.10E-11 | 0.460791 | 0.68 |
| 101 | 1 | 0.13 | 25.69 | 1.32E-45 | 0.024608 | 1.31 |
| 106b | 1 | 0.13 | 48.03 | 6.75E-28 | 0.08723 | 3.53 |
| 16 | 1 | 0.12 | 61.44 | 3.58E-26 | 0.002667 | 4.98 |
| 185 | 1 | 0.06 | 18.76 | 1.16E-39 | 0.435846 | 1.12 |
| 451 | 1 | 0.10 | 11.44 | 9.92E-21 | 0.000858 | 1.03 |
The method of calculation of normal people's mean value ask 2 for each sample at first
The normal miRNA of-Ct* 100000, the value of gained is averaged, and then 2 of each sample
The normal miRNA of-Ct* 100000 divided by mean value, and the value of gained is averaged again, all is 1 therefore.The method of calculation of carrier's mean value are at first asking 2 of each sample
-Ct carries miRNA, the value of trying to achieve is divided by normal people 2
The normal miRNA of-Ct* 100000 mean value, and then calculate mean value.The method of calculation of patient's mean value are at first asking 2 of each sample
-Ct patient miRNA, the value of trying to achieve is divided by normal people 2
The normal miRNA of-Ct* 100000 mean value, and then calculate mean value.We find out that the microRNA that conforms to Solexa result is let-7c, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p and miR-885-5p from table, that wherein, there are differences between carrier and the patient is let-7c, miR-423-5p and miR-139-5p.
Embodiment 3 is used for the making of the minuteness ribonucleic acid reagent kit of hepatitis diagnosis and prediction
The manufacture craft of minuteness ribonucleic acid reagent kit and operating process are based on solexa sequencing technologies, Realtime-PCR technology and biochip technology.
At first determine miRNA in normal human serum/blood plasma by the method or the PCR method of order-checking.By quantitative PCR technique and biochip technology screening HBVer, hepatitis B patient and the big class blood serum miRNA of normal people's expression amount difference degree, whether the index of disease and diagnosis lesion degree takes place then as prediction.The quantity of the corresponding HBVer who filters out at last and the blood serum miRNA of hepatitis B patient is controlled at tens, and this is that make on the basis in chip probe storehouse optimized simplified.This test kit comprises a collection of blood serum miRNA primer, reagent such as Taq enzyme, dNTP, wherein the primer of miRNA comprises the primer of let-7c, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p and miR-885-5p.The value of this test kit is only to need blood serum and does not need other tissue sample, detects the variation tendency of miRNA by the probe library of simplifying most, again the possibility that takes place by this variation tendency prediction hepatitis B or the pathology stage of diagnosis hepatitis B.Therefore this test kit practice of putting into production can be used in hepatitis diagnosis, treatment, therapeutic evaluation, prognosis.
Claims (4)
1. normal people, the HBVer, there are differences the miRNA combination of expression in the hepatitis B patient blood serum, it is characterized in that this combination specifically comprises let-7c, miR-1, miR-10a, miR-122, miR-125b, miR-128a, miR-128b, miR-150, miR-197, miR-221, miR-222, miR-223, miR-23a, miR-23b, miR-27a, miR-27b, miR-30a, miR-342-3p, miR-361-5p, miR-423-5p, miR-532-5p, miR-574-3p, miR-629, miR-92a, miR-92b, miR-99a, miR-139-5p, miR-193a-5p, miR-193b, miR-365, miR-375, miR-455-3p, miR-483-3p, miR-483-5p, miR-486-3p, miR-885-5p, miR-99b, let-7f, let-7g, let-7i, miR-101, miR-103, miR-106a, miR-106b, miR-107, miR-126, miR-130a, miR-130b, miR-142-3p, miR-142-5p, miR-144, miR-146a, miR-146b-5p, miR-148b, miR-151-5p, miR-15a, miR-16, miR-17, miR-182, miR-183, miR-185, miR-186, miR-18a, miR-191, miR-19b, miR-20a, miR-20b, miR-21, miR-210, miR-26a, miR-26b, miR-29c, miR-30e, miR-340, miR-362-5p, miR-363, miR-374a, miR-378, miR-424, miR-451, miR-454, miR-532-5p, miR-652, miR-660, miR-7, miR-923, miR-93, miR-96 and miR-98.
2. according to the test kit of the described miRNA combined preparation of claim 1, it is characterized in that described test kit comprises primer and Taq enzyme and the dNTP reagent of let-7c, miR-10a, miR-122, miR-125b, miR-150, miR-223, miR-23a, miR-23b, miR-27a, miR-342-3p, miR-423-5p, miR-92a, miR-99a, miR-139-5p, miR-375, miR-483-5p and miR-885-5p.
3. the preparation method of minuteness ribonucleic acid reagent kit according to claim 2 is characterized in that being made of following set step:
(1) by there are differences the miRNA of expression in the tentatively definite normal people of Solexa sequencing, HBVer, the hepatitis B patient blood serum;
(2) result according to order-checking carries out the miRNA that real time quantitative PCR method further determines to there are differences in normal people, HBVer, the hepatitis B patient blood serum expression again;
(3), finally determine can be used as the sign microRNA of difference normal people, HBVer, hepatitis B patient according to PCR result;
(4) design test kit, test kit is made of primer, the real-time quantitative PCR reagent of sign microRNA.
4. miRNA combination according to claim 1 or the application of the described miRNA composite reagent of claim 2 box in hepatitis diagnosis, treatment, prognosis.
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| CN102016037B (en) | 2013-04-10 |
| WO2010043114A1 (en) | 2010-04-22 |
| CN102016037A (en) | 2011-04-13 |
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