CN102443644A - In-situ hybridization detection kit for MICRORNA (MICRO Ribonucleic Acid)-29A level at pathologic evolution early stage of colon cancer as well as microRNA-29A in-situ hybridization detection method and application of microRNA-29a to preparation of in-situ hybridization detection kit for colon cancer - Google Patents
In-situ hybridization detection kit for MICRORNA (MICRO Ribonucleic Acid)-29A level at pathologic evolution early stage of colon cancer as well as microRNA-29A in-situ hybridization detection method and application of microRNA-29a to preparation of in-situ hybridization detection kit for colon cancer Download PDFInfo
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Abstract
The invention discloses an in-situ hybridization detection kit, which comprises a hybridization probe and a marker. The invention also discloses a method for detecting microRNA (micro Ribonucleic Acid)-29a which is closely correlated with the pathologic evolution at an early stage of colon cancer by using in-situ hybridization through the in-situ hybridization detection kit. The method comprises the following steps of: firstly, under the condition that the hybridization probe and a target sequence can form a stable hybrid complex, contacting RNA to be detected in a substrate with the hybridization probe to form a hybrid complex; and secondly, detecting the hybrid complex. According to the in-situ hybridization detection kit and the detection method disclosed by the invention, the expression of microRNA-29a can be detected in the RNA level, which is earlier than imaging medicine and traditional clinical biochemical detection index and the real RNA level screening at the early stage of canceration can be realized; and meanwhile, the detection method disclosed by the invention is simple and convenient, is low in cost and is convenience for popularization and application to county and district level hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to develop the correlation detection technology that rna expression changes (pathology evolution process) with colorectal carcinoma pathology.
Background technology
According to the data that domestic and international authoritative institution provides, the newly-increased number 2,600,000 of the annual cancer of China, death toll nearly 2,100,000; The patient more than 700 ten thousand; The annual newly-increased cancer patients 8,000,000 in the whole world, death toll is near 8,000,000, and the patient has more than 8,400 ten thousand people approximately; To double to the above number of the year two thousand twenty, this is one group of fearful numeral.Cancer diagnosis and treatment cost is increasingly high; (poor area maybe be higher by cancer patients's year medical expense 200,000; Developed regions possibly exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi; Deduction cost 35% is about 400,000,000,000, has every year 1000000000000 Renminbi to consume in vain approximately.And the cancer patients is most of can be dead soon after treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish preventative examination in advance, in time gets involved preventative regulation and control and prophylactic treatment then, accomplishes preventiveing treatment of disease of gene level cancer.
An annual report has been done by eight tame units such as U.S. sanitary research institute in 2005, cancer research institute, Disease Control and Prevention Center; Anticancer Great War to initiation in 1972 is looked back; Report thinks that the mankind are failures in anticancer Great War; Conclusion is that cancer mortality does not reduce, and it is enumerated out and causes the Several Factors of anticancer Great War failure to be: 1. tumour cell heterogeneous (polymorphum); 2. tumor cell drug resistance; 3. cancer therapy drug mentality of designing imperfection (animal model designs not science) etc.Simultaneously, also propose to examine closely again the measure of existing diagnosis and treatment cancer in this report.
The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes cancer mortality not fallen is to accomplish real early diagnosis.Come diagnosing cancer according to existing clinical medicine image (B ultrasonic, CT, zeugmatography etc.) and with other biochemistry (cancer antigen, CEACAMS, carbohydrate hormone, the cytolemma factor, the nucleus factor, cell streaming technology) index; All be that tumour forms the back diagnosis; The former will learn in a organized way and change or existing occupying lesion, latter's major part be tumour form the back secreted, discharge or the affinity tag of tumour.The clinical idea of tradition thinks that occupancy cancer piece is the diagnosis that belongs to early-stage cancer under 2 centimeters; This notion is worth conscientiously discussing, and it is rigorous inadequately that 2 centimeters early stage these of following cancer piece genus define science, analyzes from the cytology angle; 1 centimeter lump has 100,000,000 tumour cells approximately; Its three-dimensional cell stack number of 2 centimeters lump is far above 200,000,000 tumour cells, produces from canceration early stage to the mono-clonal cancer cells and forms 2 centimeters cancer piece, and its pathology evolution process is quite long; Possibly be (except the special case) more than 5 years or 10 years or even 10 years; What be difficult to confirm is in this pathology evolution process, and lump is unique spot of cancer and independent focus, and possible cancer cells is moved to other tissue or organ growth already.Clinical study confirms already, in case when forming lump, other cancer cells is moved to other position clonal growth through different approaches, in case behind the excision primary tumor, other organ recurrent foci or multiple cancer piece kitchen range successively form.Therefore; Whether define in early days with the cancerous swelling piece size below 2 centimeters clinically, rigorous inadequately (some case is when finding primary lesion; Find metastatic lesion simultaneously; Not in the content of our statement), at this moment be diagnosis in late period and treatment of late stage in fact, this is the true cause that causes cancer mortality not fallen.
Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics in order to seek more early stage diagnosing cancer, treatment cancer and preventing cancer, makes great progress.So far, we might do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level or microRNA) of gene, preceding in canceration early stage or cancer cells formation (mono-clonal), just can accomplish early prediction and examination.
MiRNA (microRNA/miRNA) is the few ribonucleic acid molecule that a segment length is about 22 nucleotides, and they can suppress translation or cause the degraded of mRNA to reduce the performance of gene by regulation and control mRNA.Many discovering arranged recently, and miRNA and cancer have the dependency of height.Therefore inference miRNA possibly be of cancerization process lead because of.Up to the present; On human body, there are 700 miRNA of surpassing to come to light; And nearly about 100 miRNA is identified with human cancer and has dependency; This wherein includes kinds of tumors such as esophagus cancer, breast cancer, leukemia, lung cancer, the cancer of the brain, liver cancer, carcinoma of the colon and rectum, glioma, pituitary tumor in esophageal cancer cell, microRNA up-regulateds in kinds of tumors such as miR-17-92 family.MiR-21 is up-regulated expression in multiple malignant tumours such as glioblastoma, mammary cancer, carcinoma of the pancreas, colorectal carcinoma, liver cancer, lung cancer, prostate cancer, cancer of the stomach.
Though tumor tissues microRNA express spectra is relevant with tumor invasion and prognosis, detection technique is complicated, wound is big, is difficult to really be applied to clinical diagnosis.Comparatively speaking, peripheral blood serum is prone to obtain and detect, and clinical application is convenient, is beneficial to popularization.Exist the express spectra of serum microRNA related in the serum with corresponding tumor tissues microRNA express spectra? So far reported the research work of multinomial serum microRNA and tumour relation in 2008, for serum microRNA provides working foundation as early diagnosis and the prognosis that molecule marker is applied to tumour.
MicroRNA can steady in a long-term exist in serum, the enzyme liberating of anti-RNA, the loss that boil, various treatment processs such as multigelation, acid or alkali environment, prolonged preservation all can not cause serum microRNA.The source of serum microRNA does not still have final conclusion, generally believes that now it derives from histiocytic active secretion process.Sophisticated microRNA is encapsulated into excision enzyme body (Exosome) by lipid or lipoprotein in cell, secrete to born of the same parents and get into blood; The excision enzyme body that gets into blood can get into recipient cell and go and encapsulate through endocytosis, discharges microRNA performance biological function.The exchange of the iuntercellular microRNA of excision enzyme body mediation is a kind of new approach of cell communication, and the stable state of keeping interior environment is played an important role.Serum microRNA mainly contains following advantage as the mark of diagnosing tumor and prognosis: the damage of detection is little, good stability, highly sensitive, can be applicable to the detection of infantile tumour.But because the expression amount of microRNA is lower in the serum, seeking a kind of detection method highly sensitive, easy and simple to handle and with low cost is that present serum microRNA is applied to clinical tumor detection problem demanding prompt solution.MicroRNA is the little RNA of non-coding of one type of high conservative; Main through the 5pUTR, coding region or the 3pUTR that combine target gene mRNA on post-transcriptional level to expression of gene row negative regulation function; The mode that microRNA participates in gene expression regulation mainly is to suppress the carrying out of translation process; Also can cause the mRNA degraded under the few cases, bioinformatic analysis shows that each microRNA possibly regulate hundreds of target genes; Prompting microRNA possibly participate in regulating signal transduction pathways numerous in the cell activities, in a series of processes such as cell proliferation, differentiation, apoptosis, immunoreation and vasculogenesis, plays a role.MiRNA is expected to become the new biomarker of diagnosing colon cancer. and normal cell and cancer cells can both discharge miRNA to peripheral blood.MiRNA in the circulation is by high stability complex body (modal is born of the same parents' ectosome and small vesica) parcel, to stop miRNA by the RNA enzyme liberating.MiR-17-3p and miR-92a obviously raise in colorectal carcinoma patient blood plasma, and behind the tumor resection tissue, these two kinds of miRNA significantly descend; MiR-92a can also with colorectal carcinoma and other diseases, differentiate like cancer of the stomach and inflammatory bowel, further analysis revealed; MiR-92a can produce 88.5% ROC area; Threshold value in RNU6B small nuclear RNA expression relatively is 240 o'clock, and the diagnostic value that its susceptibility and specificity are respectively 89% and 70%. miR-92a further is determined, and Huang etc. point out; MiR-92a and miR-29a can be from normal population discriminating colorectal cancer and high-risk adenoma, thereby confirm that miR-92a is useful to the early diagnosis colorectal carcinoma.Except peripheral blood, because colon cell constantly is shed in the enteric cavity, the specific miRNA of colorectal carcinoma is arranged also in the stool, can be used as another valuable diagnosis methods.The present invention selects many group clinical samples (cancer patient, high risk population, normal control) to adopt nucleic acid hybridization in situ technology and immunohistochemical methods method that check and analysis are carried out in the early warning of microRNA-29a and colorectal carcinoma.
The inventor finds that under study for action microRNA-29a has the obvious expression quantitative changeization colorectal carcinoma patient, cancer high risk population, normal control people, has very important clinical diagnosis meaning with microRNA-29a as the early screening colon carcinogenesis in earlier stage.MICRORNA-29A is high expression level in colon carcinogenesis process in early stage.He do in examination in canceration early stage, and treatment of colon cancer after recurrence, shift early warning very important clinical meaning also arranged.
The contriver is in long term studies; Drawn a kind of new concept, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, and can not only stop present treating the disease affected (morbidity back diagnosis and treatment); Accomplish preventative diagnosis and treatment; Accomplish treating the disease affected, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, the contriver innovates theoretical and technical in the rna level kit for screening and medicine of exploitation and production major disease.Particularly screen clinical samples (normal population, cancer high risk population, tumour patient); Broken through healthy tissues and tumor tissues consistency research and development thinking relatively, sought and developed the rna level that becomes before the cancer, developed closely related with cancer early gene physiopathology; And the extremely important target of clinical meaning; Tumour is clinically formed the preventative diagnosis and treatment that diagnosis and treatment pattern in back becomes tumour, striven for the time and the space of tumor diagnosis and treatment, reach preventing cancer.
At present; MICRORNA-29A express spectra detection method is analyzed technology with Northern hybridization, expression chip, real-time fluorescence quantitative PCR and Solexa order-checking etc.; And these methods are used for the scientific research aspect more, the incompatibility clinical application, and detect RNA than genetic analysis more science (the DNA analysis major part is on the presentation of susceptibility; RNA is functional embodiment), than analysis of protein more reliable (RNA and albumen are transcribed sometimes asynchronous).The detection technique and the test kit that adopt hybridization in situ technique and groupization immunization method to detect MicroRNA-29a horizontal expression amount according to the existing literature data do not appear in the newspapers.
The inventor is in the requirement to the novelty invention; Designed (colorectal carcinoma patient, high risk population, normal control) different pieces of information example group; Detect with hybridization in situ technique; The result shows above colon cancer patient MICRORNA-29A high expression level, and the high risk population has and expresses 15-25% in various degree, and normal control all is low the expression.The important symbol thing that shows the examination in early stage of MICRORNA-29A colon carcinogenesis.
Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark; Under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized; With radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is molecule or sequence the unknown but the known nucleic acid molecule of molecule (though indeterminate this molecule full sequence of known array; But what target molecule known its is directed against), the kind of probe can be divided into dna probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe again by the properties of nucleic acids difference.For the ease of spike, probe must be used certain means mark in addition, is beneficial to later detection.Affinity tag commonly used comprises radionuclide and non-radioactive marker's two big classes.Isotopic label commonly used has
3H,
35S,
125I with
32P.Advantages such as susceptibility is high though isotopic label has, the back of the body end is comparatively clear because ri all can damage human and environment, have by the substituted trend of heterotope recently.At present the most frequently used in the heterotope affinity tag have three kinds of vitamin H, digoxin and resorcinolphthaleins.The method that detects these affinity tags all is a sensitive extremely.
According to used probe and to detect nucleic acid difference can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization.No matter but the hybridization of any form, all must be through five big processes, promptly histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the hybridization mode of RNA-RNA, and synthetic probe (RNA) and the target RNA that detects are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research with observe, start and termination place the result not influence of residue to detecting.
In view of the diagnosis of cancer clinically (medical imaging and biochemical indicator thing all are the diagnosis after tumour forms) at present is the diagnosis in late period, treatment also is a treatment of late stage, the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the diagnosis and treatment pattern of major disease clinically; Become preventative preventiveing treatment of disease from treating the disease affected; Reach preventative diagnosis and treatment; Present medical imaging means and numerous biochemical marker can't be detected become rna level before the cancer and quantize changes technology, do the technological breakthrough of novelty, provide that to become the rna level examination before the cancer technological.Making has had a preceding technology that becomes the real early screening of rna level of new cancer clinically, for the diagnosis and treatment of clinical cancer are raced against time and the space.
Summary of the invention
The object of the invention at first provides a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and affinity tag.Secondly, the present invention also will provide the mentioned reagent box to be used for shifting the relevant in situ hybridization detection method of early warning with colon carcinogenesis examination in early stage and treatment back.
For realizing the object of the invention; Technical scheme of the present invention is following: the present invention at first provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and affinity tag, wherein; Described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1; Sequence number: NR_029503.1, nucleotide sequence length is 64bp, is positioned at karyomit(e) 7q32.3 " on.
A preferred version of test kit of the present invention is that described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Canceration of the present invention MICRORNA-29A kit for screening using value in earlier stage is, to various cancerations examinations in early stage, reaches after the canceration or after the treatment and recurs, shifts, spreads early warning takes place, and further cooperates clinical treatment.
The present invention also provides a kind of detection method of MICRORNA-29A in situ hybridization, may further comprise the steps:
(1) described in the above hybridization probe and target sequence can form under the condition of stablizing hybridization complex, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said hybridization complex.
Detection method of the present invention, wherein preferably, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood leucocyte sample or other organ-tissue cell specimen for use.More preferably be that described blood preparation or other organ-tissue cell specimen are from colorectal carcinoma patient, colorectal carcinoma high risk population, healthy normal population.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine; With MICRORNA-29A is detected object; Synthesising probing needle is the complementary sequence of MICRORNA-29A sequence, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic RNA.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of MICRORNA-29A.The expression amount of above RNA is judged in colour developing according to hybridization back immunohistochemical methods, and normal people MICRORNA-29A is low to express, i.e. colour developing on a small quantity, and MICRORNA-29A has apparent difference at colon cancer patient and normal control, and this expression of gene amount is all higher than normal people expression amount.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, adds Digestive system automatically;
3). instrument discards liquid automatically, and the back is fixing automatically;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, cleans automatically;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, cleans automatically;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, cleans colour developing automatically;
10). take out the mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with MICRORNA-29A synthetic nucleic probe with digoxigenin labeled (cDNA of digoxigenin labeled, RNA and oligonucleotide probe; Not only probe has a biotin labeling advantage; Also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position); This hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized,, under light microscopic, observe existence and the location of RNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of purpose RNA.
The inventive method is a nucleic acid hybridization in situ technology commonly used at present; This method is through detecting the MICRORNA-29A expression amount in the substrate cell; Be used for confirming that colorectal carcinoma pathology develops the RNA variable quantity in early stage, the various cancerations of early warning whether take place and the colorectal carcinoma patient treatment after the prediction of whether recurring, shifting.Because MICRORNA-29A is low the expression in the normal people, if the MICRORNA-29A expression amount increases, the risk of suffering from cancer be described, explain that canceration takes place, or the cancer patient postoperative recurs, shifts, thus the diagnostic message of acquisition cancer.A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect:
Clinical meaning of the present invention is the more early stage variation that detects MICRORNA-29A expression amount in various canceration generations and the pathology evolution process of following the tracks of, and the early warning colorectal carcinoma takes place, development trend.Diagnostic kit of the present invention and other detection and cancer markers clinically, and the medical imaging inspection has apparent difference.The present invention can detect the MICRORNA-29A unconventionality expression at rna level; Before the recurrence of occupancy carninomatosis kitchen range is not found in the medical imaging inspection; Before the cancer biochemical indicator does not produce unusually; Also do not form before the tumour, can accomplish the information acquisition of above abnormal gene expression early, predict for real early warning of clinical carninomatosis patient and treatment back transfer and relapse early.So just might implement early screening, early prevention, the early treatment of cancer, might from the source, thoroughly effect a radical cure the colorectal carcinoma foul disease.
In addition, characteristics highly sensitive, high specificity that test kit provided by the invention has, simultaneously, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Description of drawings
Fig. 1 is a MICRORNA-29A hybridization in situ technique schema of the present invention.
Fig. 2 is that colon cancer patient MICRORNA-29A expresses picture in the embodiment of the invention.
Fig. 3 is high risk population's picture.
Fig. 4 is that normal people MICRORNA-29A expresses picture in the embodiment of the invention.
Embodiment
Below in conjunction with embodiment, content of the present invention is described more specifically.Should be appreciated that following embodiment is used for explanation and non-limiting content of the present invention, any pro forma change or accommodation will fall into protection scope of the present invention.
Embodiment 1
Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises that wherein: the probe mark thing of present embodiment is selected digoxin for use with hybridization probe, affinity tag, the specification sheets of MICRORNA-29A design.
The test kit hybridization solution is formed:
| Digestive system | 100 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| Protection liquid | 100 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| Prehybridization solution | 1300 μ L/ pipe | 2 pipe/boxes | Colourless transparent liquid |
| The justice hybridization solution | 10 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| The antisense hybridization solution | 10 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| Confining liquid | 1000 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| The alkaline phosphatase enzyme antibody | 1 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| Developer A | 175 μ L/ pipe | 1 pipe/box | Yellow liquid |
| Developer B | 320 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
| The damping fluid I | The 90mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid II | The 80mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid III | The 20mL/ bottle | 3 bottle/boxes | Light yellow or colourless transparent liquid |
| The damping fluid IV | The 90mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
| Stationary liquid | The 90mL/ bottle | 1 bottle/box | Colourless transparent liquid |
| The positive control sample | 6/box | ? | ? |
The reagent preparation working concentration
1). 10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;
2). 20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;
Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;
3). 10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;
4) .10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * the damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).
Embodiment 2
Use the implementation process of nucleic acid hybridization in situ detection method to each group blood preparation MICRORNA-29A expression amount:
1). get two of samples to be measured;
2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 * damping fluid I 99.9ml, are working concentration) 50 ml, 37 ℃ of water-bath preheating 10min put 16 slides into, handle 12 min, use 1 * damping fluid I to wash 5min again for 37 ℃;
3). the protection liquid with 0.2% (protection liquid 1ml adds 1 * damping fluid I, and 99ml is working concentration) is washed 10min, and tri-distilled water is washed 5min (above process is all carried out at glass jar), takes out slide, lets its seasoning;
4). slide is put into the box of preserving moisture, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;
5). take out slide, discard deckglass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into the box of preserving moisture, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered is covered the box of tightly preserving moisture, and is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard deckglass, slide is put into glass jar:
In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II;
In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II;
In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;
8). wash 30s with 1 * damping fluid III, take out slide, seasoning;
9). slide is put into the box of preserving moisture, add 0.5% confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III), 100 μ L/ sheets, cover the box of tightly preserving moisture, at room temperature act on 30min.(this step need not add deckglass);
10). take out slide, wash 30s with 1 * damping fluid III, seasoning;
11). slide is put into the box of preserving moisture; Add X-AP antibody (getting a pipe alkaline phosphatase enzyme antibody) 100 μ L/ sheets, cover the box of tightly preserving moisture and at room temperature act on 30min to wherein adding 1.8ml 1 * damping fluid III; Time can not be long, otherwise can produce false positive (this step need not add deckglass);
12). take out slide, wash 3 times, each 15min with 1 * damping fluid III;
13). wash 2min with 1 * damping fluid IV, add developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL 1 * damping fluid IV, mixing), room temperature lucifuge 16h is to more than the 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is used digoxigenin labeled with goal gene; Become the RNA nucleic probe; The RNA nucleic acid to be measured of probe and human leukocytes is hybridized,, therefore under light microscopic, observe existence and the location of RNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of purpose RNA.
20 of colon cancer patients, 20 of high risk population's (polyp intestinal), 20 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result representes that all cancer patients MICRORNA-29A expression amounts are high, and cell dyeing is dark; The high risk population expresses slightly and reduces, decimal dyeing; Normal control group MICRORNA-29A expression amount is low, the dyeing of cell minority, and concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
| Intestinal cancer disease number | Expression amount % | High-risk number | Expression amount % | Normal number | Expression amount % |
| 1 | 80 | 1 | 28 | 1 | 0 |
| 2 | 74 | 2 | 20 | 2 | 0 |
| 3 | 62 | 3 | 22 | 3 | 0 |
| 4 | 64 | 4 | 18 | 4 | 4 |
| 5 | 72 | 5 | 26 | 5 | ? ?0 |
| 6 | 82 | 6 | 20 | 6 | 0 |
| 7 | 78 | 7 | 22 | 7 | 0 |
| 8 | 82 | 8 | 28 | 8 | 2 |
| 9 | 70 | 9 | 26 | 9 | 5 |
| ? 10 | 84 | ? 10 | 22 | ? 10 | 0 |
| ? 11 | 72 | ? 11 | 22 | ? 11 | 2 |
| 12 | 78 | ? 12 | 24 | ? 12 | 0 |
| 13 | 80 | ? 13 | 16 | ? 13 | 3 |
| 14 | 74 | ? 14 | 18 | ? 14 | 0 |
| ? 15 | 68 | ? 15 | 22 | ? 15 | 0 |
| ? 16 | 80 | ? 16 | 24 | ? 16 | 0 |
| ? 17 | 78 | ? 17 | 26 | ? 17 | 0 |
| ? 18 | 70 | ? 18 | 16 | ? 18 | 0 |
| ? 19 | 82 | ? 19 | 14 | ? 19 | 0 |
| ? 20 | 76 | 20 | 23 | 20 | 2 |
。
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Claims (10)
1. a hybridization in situ detection kit comprises hybridization probe and affinity tag, it is characterized in that, described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1.
2. test kit as claimed in claim 1 is characterized in that, described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.
3. test kit as claimed in claim 1 is characterized in that this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1 is characterized in that this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1 is characterized in that this test kit also comprises developer.
6. microRNA-29A gene hybridization in situ detection method is characterized in that this method may further comprise the steps:
(1) can form under the condition of stablizing hybridization complex at described hybridization probe of claim 1 and target sequence, RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said hybridization complex.
7. detection method as claimed in claim 6 is characterized in that, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6 is characterized in that, described substrate is selected people's blood leucocyte sample for use.
9. detection method as claimed in claim 8 is characterized in that, described blood leucocyte sample is selected from cancer, high risk population, normal people's sample.
10.microRNA-29A gene detects the application in the colorectal carcinoma pathology in situ hybridization test kit in preparation, it is characterized in that said test kit contains the described hybridization probe of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104290796A CN102443644A (en) | 2011-12-20 | 2011-12-20 | In-situ hybridization detection kit for MICRORNA (MICRO Ribonucleic Acid)-29A level at pathologic evolution early stage of colon cancer as well as microRNA-29A in-situ hybridization detection method and application of microRNA-29a to preparation of in-situ hybridization detection kit for colon cancer |
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| CN110229870A (en) * | 2019-04-04 | 2019-09-13 | 北京化工大学 | A kind of intracellular miRNA quantitative approach based on single molecular fluorescence imaging |
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| CN100999765A (en) * | 2006-12-20 | 2007-07-18 | 南京大学 | Biological chip of identifying colon canceration degree using microchanging of RNA |
| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
| CN101657547A (en) * | 2006-07-13 | 2010-02-24 | 俄亥俄州立大学研究基金会 | Be used to diagnose and treat the method and composition based on Microrna of colon cancer-related diseases |
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| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
| CN101657547A (en) * | 2006-07-13 | 2010-02-24 | 俄亥俄州立大学研究基金会 | Be used to diagnose and treat the method and composition based on Microrna of colon cancer-related diseases |
| CN100999765A (en) * | 2006-12-20 | 2007-07-18 | 南京大学 | Biological chip of identifying colon canceration degree using microchanging of RNA |
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