CN102605050A - Assay kit and method of mRNA (messenger ribonucleic acid) level of drug resistance gene (FBW7) through in situ hybridization before chemotherapy of various cancers and application of FBW7 gene - Google Patents
Assay kit and method of mRNA (messenger ribonucleic acid) level of drug resistance gene (FBW7) through in situ hybridization before chemotherapy of various cancers and application of FBW7 gene Download PDFInfo
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- CN102605050A CN102605050A CN2011104429953A CN201110442995A CN102605050A CN 102605050 A CN102605050 A CN 102605050A CN 2011104429953 A CN2011104429953 A CN 2011104429953A CN 201110442995 A CN201110442995 A CN 201110442995A CN 102605050 A CN102605050 A CN 102605050A
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Abstract
The invention discloses an in situ hybridization assay kit. The kit comprises a hybridization probe and a label. The invention also discloses a method for assaying mRNA (messenger ribonucleic acid), a primary transcript of a tumor-inhibiting factor FBW7 gene closely related to cancer chemotherapy drug resistance through in situ hybridization by using the kit. The method comprises the following steps: (1) under the condition that the hybridization probe and a target sequence can form a stable hybrid complex, and contacting the RNA to be assayed in a substrate with the hybridization probe to form the hybrid complex; and (2) assaying the hybrid complex. The kit and the method have the following beneficial effects: the expression of the FBW7 gene can be assayed on the mRNA level, so the clinicians can be guided to implement effective treatment on the cancer patients; and meanwhile, the method is simple and convenient, is low in cost and is convenient to popularize and apply in the county/district-level hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to express the correlation detection technology that changes with cancer drug resistant gene mRNA.
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.
Resistance problem in the disease drug treatment is a more serious clinical problem.At present, have personalized gene SNP catastrophe point to detect clinically, this can find some and drug resistance related gene not enough, makes the reason (finding to be only general orientation with the gene of disease-related) of sudden change clear.It is the relevant gene of tumor drug resistance mechanism that report FBW7 is arranged.Chemotherapy is one of the most important means of malignant tumour of treating clinically at present, yet causes the patient no longer responsive to treatment owing to tumour cell usually can produce resistance to chemotherapeutics, finally causes chemotherapy failure even palindromia.Scientists has been carried out number of research projects to tumor drug resistance mechanism in recent years, and result of study shows that tumor drug resistance and ingestion of medicines reduce, and discharge increases; Activation reduces; Number of mechanisms such as inactivation increases, and the dna damage reparation increases, and dna methylation and signal transduction pathway are unusual are relevant." nature " of up-to-date first phase (Nature) on the magazine two research groups the resistance mechanism of tumour has been pointed to same gene---FBW7.This discovers and helps the oncologist to predict the drug reaction that patients undergoing chemotherapy will produce taxol and similar active medicine, and has indicated new research target spot for cancer therapy.Taxol is a most widely used clinically at present based chemotherapy medicine.Taxol can cause that a large amount of microtubules gather in the cell through promoting microtubule polymerization and stable polymerization microtubule, thus the various functions of resultant interference cell, make cell fission stop at mitotic division its, the proper splitting of blocking-up cell.Big quantity research shows that taxol has the curative effect of wide spectrum for kinds of tumors such as ovarian cancer, mammary cancer, lung cancer, large bowel cancer, melanoma, Head and Neck cancer, lymphoma, brain tumors.Yet clinical in recent years also have many case report patients after accepting paclitaxel treatment, to show drug resistance, finally causes the treatment failure.In first piece of paper, Wertz and colleague find to accept short survivin MCL1 in the reacting cells after taxol and the another kind of anti-microtubule medicine vincristine(VCR) treatment and are showing and reduce.In further studying, they confirm that a kind of known TIF FBW7 has brought into play critical effect in the MCL1 proteolytic degradation of uiquitin-protease enzyme body mediation.The research in past confirms that the FBW7 defective comprises that with multiple cancer mammary cancer is relevant with colorectal carcinoma.Wertz etc. observe in ovarian cancer and the colon cancer cell of FBW7 defective and express high-caliber MCL1 albumen, and are easier to resist the microtubule medicine and produce tolerance.In another piece paper; Study to the effect of FBW7 in T cell acute lymphoblastic leukemia (T-ALL) cell from U.S. Bai Si Israel Di Kenneth medical center (Beth Israel Deaconess Medical Center) Wenyi Wei and colleague, Jun, Myc and Jadded1 1 albumen are high level expression in the T-ALL cell of discovery FBW7 defective.These albumen high expression levels are understood inducing cell generation apoptosis usually under the normal circumstances, and the T-ALL cell of FBW7 defective does not show this effect.Wenyi Wei is further obtaining the research conclusion the same with Wertz in the research with the colleague: under the proteic situation of disappearance FBW7, and the cell MCL1 that can't degrade, thereby the apoptosis that made cells escape.In addition, Wenyi Wei and his colleague have also confirmed getting in touch between FBW7 and the tumour medicine tolerance.In experiment, Wenyi Wei etc. attempt handling with a kind of experimental drug the T-ALL cell of FBW7 defective, find that cell is insensitive to medicine.And after they utilize Suo Lafeini to reduce the MCL1 level in the cell, find that these cells have recovered susceptibility to experimental drug.Rider not. the oncologist of Hutchinson DKFZ and molecular biologist Bruce Clurman make comments to this result of study and claim that this is to make us the discovery of feeling excited, also only are PRELIMINARY RESULTS but he stresses to obtain at present simultaneously.He points out that FWB7 all has damage effect to the multiple protein in the tumour cell, and is not only MCL1." when you upset FBW7, be difficult to confirm what kind of effect which target molecule in downstream has brought into play in tumour takes place.These two the important concentrated regulating and controlling effects of FBW7 of having studied of research to MCL1, this might also have certain distance from complete story, " Bruce Clurman says.The knubble biological scholar Hayley McDaid suggestion of Alberta einstein medical college is research taxanes pharmacological agent cancer patients's archives further.She thinks that if the conclusion of Wertz etc. is set up really the researchist should endeavour to probe into the mutual relationship between FBW7 and the taxol reaction." these samples are done some sequential analyses be necessary, " Hayley McDaid says
The mRNA of the FBW7 gene early screening before as the medication of cancer patients's chemotherapy there is very important clinical diagnosis meaning.The mRNA of FBW7 is low the expression or zero expression in lung cancer and mammary cancer chemotherapy resistance patient, and screening plays an important role ability direct clinical doctor to chemotherapeutics.
At present high flux gene chip, Northern Blot, proteinogram analytical technology are all adopted in the research of FBW7 gene; And these methods are used for the scientific research aspect more; The incompatibility clinical application; And detect mRNA than genetic analysis science (the DNA analysis major part is on the presentation of susceptibility, and mRNA is functional embodiment) more, than analysis of protein more reliable (mRNA and albumen are transcribed sometimes asynchronous).Detection technique and test kit according to existing literature data FBW7 gene mRNA level do not appear in the newspapers.
The inventor has adopted with hybridization in situ technique and has detected in the requirement to the novelty invention, and the result shows lung cancer and low expression of mammary cancer resistance patient FBW7 gene or zero the expression, shows that the FBW7 gene is the cancer drug resistance related gene.
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; Synthetic probe (mRNA) and the said target mrna that detects are the principles that adopts base complementrity (hybridization is complementary); Simultaneously through long-time research and observation; Start and termination place the result not influence (because mRNA sequence that contriver adopt all surpass 600bp more than) of residue to detecting.
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 the in situ hybridization detection method with cancer patients's chemotherapy resistance genes involved.
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 a sequence shown in the SEQ ID NO.1, is to get one section sequence in the FBW7 gene, obtains from 200 to 1000bp; FBW7 gene order number: AY033553; Be sequence shown in the SEQ ID NO.2, the nucleotide sequence length of gene is 2063bp, and CDS is 97 ... 1980bp.If (gene order is oversize in the probe mark process; Surpass more than the 1000bp, we use the sequence of CDS and come designing probe, if the sequence of CDS also surpasses more than the 1000bp; Can adopt one section base sequence in centre of gene to come synthesising probing needle; Base sequence is no less than 500bp, will do sequential detection after probe is synthetic, and function is carried out the analysis of clinical meaning).
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 FBW7 gene screening in early stage test kit using value of the present invention is, can drug resistant gene before cancer patients's chemotherapy be detected in the mRNA level, further cooperates and guiding clinical treatment.
The present invention also provides a kind of detection method of FBW7 gene hybridization in situ, 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 the cancer patients.
Detection method of the present invention wherein preferably, detects sample before described clinical all kinds of cancer patients's chemotherapy.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine; One-level functional transcription product mRNA with the FBW7 gene is a detected object; Synthesising probing needle is the RNA sequence of FBW7 gene, 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 the sxemiquantitative or the quantitative expression deciding degree of FBW7 gene.Above expression of gene amount is judged in colour developing according to hybridization back immunohistochemical methods, and lung cancer and mammary cancer patient FBW7 gene are low expresses, i.e. colour developing or do not develop the color in a small amount, and the FBW7 gene is all low at cancer resistance patient expression amount, most of resistance patient zero expression.At normal population is high expression level, a large amount of cell dyeings.
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 the FBW7 gene is that goal gene synthetic nucleic probe is 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 mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.
The inventive method is a nucleic acid hybridization in situ technology commonly used at present, and this method is used for observing the mRNA variable quantity of cancer patients's drug resistant gene through detecting the FBW7 gene expression amount in the substrate cell, prediction cancer patients resistance situation.Because FBW7 gene high expression level in the normal people if cancer patients FBW7 gene expression amount is low, explains that the patient has resistance, thereby obtain the drug-fast information of cancer patients.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 that chemotherapy detects drug resistant gene in earlier stage, can the medication of direct clinical doctor chemotherapy.
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 FBW7 gene hybridization in situ techniqueflow chart of the present invention.
Fig. 2 is that lung cancer patient FBW7 expresses the reduction picture in the embodiment of the invention.
Fig. 3 is that mammary cancer patient FBW7 expresses the reduction picture in the embodiment of the invention.
Fig. 4 is that normal people FBW7 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.
Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises with the FBW7 gene being hybridization probe, affinity tag, the specification sheets of testing goal gene design, wherein:
The probe mark thing of present embodiment is selected digoxin for use.
The test kit hybridization solution is formed:
| Digestive system | 100 μ L/ | 1 pipe/box | Colourless transparent liquid |
| Protection liquid | 100 μ L/ | 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/ | 1 pipe/box | Colourless transparent liquid |
| The antisense hybridization solution | 10 μ L/ | 1 pipe/box | Colourless transparent liquid |
| Confining liquid | 1000 μ L/ | 1 pipe/box | Colourless transparent liquid |
| The alkaline | 1 μ L/ | 1 pipe/box | Colourless transparent liquid |
| Developer A | 175 μ L/ | 1 pipe/box | Yellow liquid |
| Developer B | 320 μ L/ | 1 pipe/box | Colourless transparent liquid |
| The damping fluid I | The 90mL/ | 1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid II | The 80mL/ | 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/ | 1 bottle/box | Light yellow or colourless transparent liquid |
| Stationary liquid | The 90mL/ | 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 FBW7 gene 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). (protection liquid 1ml adds 1 * damping fluid
to the protection liquid with 0.2%; 99ml is working concentration) wash 10min; Tri-distilled water is washed 5min (above process is all carried out at glass jar); Take out slide, let 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 mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.
20 of lung cancer patients, 20 of mammary cancer, 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 FBW7 gene expression amounts are low, and the cell majority does not dye, and concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
| The lung cancer number | Expression amount % | The mammary cancer number | Expression amount % | Normal number | |
| 1 | 0 | 1 | ?0 | 1 | 72 |
| 2 | 0 ? | 2 | 2 | 2 | 76 |
| 3 | ? 0 | 3 | 0 | 3 | 79 |
| 4 | 0 ? | 4 | 0? ? | 4 | 86? |
| 5 | ? 2 | 5 | 0 | 5 | 78 |
| 6 | 0 ? | 6 | 2 | 6 | 86 |
| 7 | ? 0 | 7 | 0 | 7 | 60 |
| 8 | ? 0 | 8 | 0 | 8 | 68 |
| 9 | ? 2 | 9 | 0 | 9 | 82 |
| ? 10 | ? 0 | ? 10 | 0 | ? 10 | 84 |
| ? 11 | ? 0 | ? 11 | 2 | ? 11 | 72 |
| ? 12 | ? 3 | 12 | 0 | ? 12 | 80 |
| ? 13 | ? 0 | ? 13 | 0 | ? 13 | 76 |
| ? 14 | ? 0 | ? 14 | 2 | ? 14 | 80 |
| ? 15 | ? 2 | ? 15 | 2 | ? 15 | 79 |
| ? 16 | ? 0 | ? 16 | 0 | ? 16 | 76 |
| ? 17 | ? 0 | ? 17 | 0 | ? 17 | 74 |
| ? 18 | ? 0 | ? 18 | 2 | ? 18 | 78 |
| ? 19 | ? 0 | ? 19 | 0 | ? 19 | 82 |
| 20 | 2 | 20 | 0 | 20 | 87 |
。
Sequence table
SEQ ID NO:1 (probe sequence)
200?catgtccaca?ttagaatctg?tgacatacct?acctgaaaaa
241?ggtttatatt?gtcagagact?gccaagcagc?cggacacacg?ggggcacaga?atcactgaag
301?gggaaaaata?cagaaaatat?gggtttctac?ggcacattaa?aaatgatttt?ttacaaaatg
361?aaaagaaagt?tggaccatgg?ttctgaggtc?cgctcttttt?ctttgggaaa?gaaaccatgc
421?aaagtctcag?aatatacaag?taccactggg?cttgtaccat?gttcagcaac?accaacaact
481?tttggggacc?tcagagcagc?caatggccaa?gggcaacaac?gacgccgaat?tacatctgtc
541?cagccaccta?caggcctcca?ggaatggcta?aaaatgtttc?agagctggag?tggaccagag
601?aaattgcttg?ctttagatga?actcattgat?agttgtgaac?caacacaagt?aaaacatatg
661?atgcaagtga?tagaacccca?gtttcaacga?gacttcattt?cattgctccc?taaagagttg
721?gcactctatg?tgctttcatt?cctggaaccc?aaagacctgc?tacaagcagc?tcagacatgt
781?cgctactgga?gaattttggc?tgaagacaac?cttctctgga?gagagaaatg?caaagaagag
841?gggattgatg?aaccattgca?catcaagaga?agaaaagtaa?taaaaccagg?tttcatacac
901?agtccatgga?aaagtgcata?catcagacag?cacagaattg?atactaactg?gaggcgagga
961?gaactcaaat?ctcctaaggt?gctgaaagga?catgatgatc?atgtgatcac
SEQ ID NO:2 FBW7 gene order number: the AY033553 gene order is following:
1?agtcaaggct?ttgacagggc?atagtctcct?ccaataatct?tctccgttct?ctctcattat
61?tccctcgagt?tcttctcagt?caagctgcat?gtatgtatgt?gtgtcccgag?aagcggtttg
121?atactgagct?gcatttgcct?ttactgtgga?gttttgttgc?cggttctgct?ccctaatctt
181?ccttttctga?cgtgcctgag?catgtccaca?ttagaatctg?tgacatacct?acctgaaaaa
241?ggtttatatt?gtcagagact?gccaagcagc?cggacacacg?ggggcacaga?atcactgaag
301?gggaaaaata?cagaaaatat?gggtttctac?ggcacattaa?aaatgatttt?ttacaaaatg
361?aaaagaaagt?tggaccatgg?ttctgaggtc?cgctcttttt?ctttgggaaa?gaaaccatgc
421?aaagtctcag?aatatacaag?taccactggg?cttgtaccat?gttcagcaac?accaacaact
481?tttggggacc?tcagagcagc?caatggccaa?gggcaacaac?gacgccgaat?tacatctgtc
541?cagccaccta?caggcctcca?ggaatggcta?aaaatgtttc?agagctggag?tggaccagag
601?aaattgcttg?ctttagatga?actcattgat?agttgtgaac?caacacaagt?aaaacatatg
661?atgcaagtga?tagaacccca?gtttcaacga?gacttcattt?cattgctccc?taaagagttg
721?gcactctatg?tgctttcatt?cctggaaccc?aaagacctgc?tacaagcagc?tcagacatgt
781?cgctactgga?gaattttggc?tgaagacaac?cttctctgga?gagagaaatg?caaagaagag
841?gggattgatg?aaccattgca?catcaagaga?agaaaagtaa?taaaaccagg?tttcatacac
901?agtccatgga?aaagtgcata?catcagacag?cacagaattg?atactaactg?gaggcgagga
961?gaactcaaat?ctcctaaggt?gctgaaagga?catgatgatc?atgtgatcac?atgcttacag
1021?ttttgtggta?accgaatagt?tagtggttct?gatgacaaca?ctttaaaagt?ttggtcagca
1081?gtcacaggca?aatgtctgag?aacattagtg?ggacatacag?gtggagtatg?gtcatcacaa
1141?atgagagaca?acatcatcat?tagtggatct?acagatcgga?cactcaaagt?gtggaatgca
1201?gagactggag?aatgtataca?caccttatat?gggcatactt?ccactgtgcg?ttgtatgcat
1261?cttcatgaaa?aaagagttgt?tagcggttct?cgagatgcca?ctcttagggt?ttgggatatt
1321?gagacaggcc?agtgtttaca?tgttttgatg?ggtcatgttg?cagcagtccg?ctgtgttcaa
1381?tatgatggca?ggagggttgt?tagtggagca?tatgatttta?tggtaaaggt?gtgggatcca
1441?gagactgaaa?cctgtctaca?cacgttgcag?gggcatacta?atagagtcta?ttcattacag
1501?tttgatggta?tccatgtggt?gagtggatct?cttgatacat?caatccgtgt?ttgggatgtg
1561?gagacaggga?attgcattca?cacgttaaca?gggcaccagt?cgttaacaag?tggaatggaa
1621?ctcaaagaca?atattcttgt?ctctgggaat?gcagattcta?cagttaaaat?ctgggatatc
1681?aaaacaggac?agtgtttaca?aacattgcaa?ggtcccaaca?agcatcagag?tgctgtgacc
1741?tgtttacagt?tcaacaagaa?ctttgtaatt?accagctcag?atgatggaac?tgtaaaacta
1801?tgggacttga?aaacgggtga?atttattcga?aacctagtca?cattggagag?tggggggagt
1861?gggggagttg?tgtggcggat?cagagcctca?aacacaaagc?tggtgtgtgc?agttgggagt
1921?cggaatggga?ctgaagaaac?caagctgctg?gtgctggact?ttgatgtgga?catgaagtga
1981?agagcagaaa?agatgaattt?gtccaattgt?gtagacgata?tactccctgc?ccttccccct
2041?gcaaaaagaa?aaaaaaaaaa?aaa
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 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. FBW7 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 the cancer patients.
10.FBW7 gene detects the application in the various cancerous lesion in situ hybridization test kits in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104429953A CN102605050A (en) | 2011-12-27 | 2011-12-27 | Assay kit and method of mRNA (messenger ribonucleic acid) level of drug resistance gene (FBW7) through in situ hybridization before chemotherapy of various cancers and application of FBW7 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104429953A CN102605050A (en) | 2011-12-27 | 2011-12-27 | Assay kit and method of mRNA (messenger ribonucleic acid) level of drug resistance gene (FBW7) through in situ hybridization before chemotherapy of various cancers and application of FBW7 gene |
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| Publication Number | Publication Date |
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| CN102605050A true CN102605050A (en) | 2012-07-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011104429953A Pending CN102605050A (en) | 2011-12-27 | 2011-12-27 | Assay kit and method of mRNA (messenger ribonucleic acid) level of drug resistance gene (FBW7) through in situ hybridization before chemotherapy of various cancers and application of FBW7 gene |
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| CN (1) | CN102605050A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114959044A (en) * | 2022-06-29 | 2022-08-30 | 华中农业大学 | Pi-FISH + nucleic acid probe set, in-situ hybridization method and application thereof |
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| CN1281508A (en) * | 1997-12-19 | 2001-01-24 | 法玛西雅厄普约翰美国公司 | Human Sel-10 polypeptides and polynucleotides that encode them |
| WO2008153743A2 (en) * | 2007-05-21 | 2008-12-18 | Dana Farber Cancer Institute | Compositions and methods for cancer gene discovery |
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| CN1281508A (en) * | 1997-12-19 | 2001-01-24 | 法玛西雅厄普约翰美国公司 | Human Sel-10 polypeptides and polynucleotides that encode them |
| WO2008153743A2 (en) * | 2007-05-21 | 2008-12-18 | Dana Farber Cancer Institute | Compositions and methods for cancer gene discovery |
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| GENEBANK: "AY033553", 《GENEBANK》, 22 August 2001 (2001-08-22) * |
| 郭晓强等: "泛素连接酶FBW7的肿瘤抑制作用和机制", 《中国生物化学与分子生物学报》, 20 November 2011 (2011-11-20), pages 998 - 1006 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114959044A (en) * | 2022-06-29 | 2022-08-30 | 华中农业大学 | Pi-FISH + nucleic acid probe set, in-situ hybridization method and application thereof |
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Application publication date: 20120725 |