CN103882099A - LKB1(Liver Kinase B1)mRNA (messenger RNA) detection method, kit and gene chip - Google Patents

LKB1(Liver Kinase B1)mRNA (messenger RNA) detection method, kit and gene chip Download PDF

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Publication number
CN103882099A
CN103882099A CN201210562544.8A CN201210562544A CN103882099A CN 103882099 A CN103882099 A CN 103882099A CN 201210562544 A CN201210562544 A CN 201210562544A CN 103882099 A CN103882099 A CN 103882099A
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lkb1mrna
situ hybridization
quantum dot
hybridization probe
cooh
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万晓春
王伟
粟武
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Abstract

The invention discloses a LKB1(Liver Kinase B1)mRNA (messenger RNA) detection method which comprises the following steps: preparing a LKB1mRNA in-situ hybridization probe; using quantum dots to mark the LKB1mRNA in-situ hybridization probe to obtain a quantum-dot LKB1mRNA in-situ hybridization probe; using the quantum-dot LKB1mRNA in-situ hybridization probe for hybridizing in situ with a specimen; and placing the hybridized specimen under a microscope for observation. The invention also provides a kit and a gene chip which comprise the quantum-dot LKB1mRNA in-situ hybridization probe. According to the detection method, the quantum-dot LKB1mRNA in-situ hybridization probe is used, the toxicity of the quantum dots is less than that of a fluorescent dye, and the quantum dots are more secure to use, a colored specimen can be stored for a long time for repeated observation, and the detection method has the advantages of simple operation and high sensitivity.

Description

A kind of LKB1mRNA detection method, test kit and gene chip
Technical field
The present invention relates to biological technical field, be specifically related to a kind of LKB1mRNA detection method, test kit and gene chip.
Background technology
People LKB1(Liver Kinase B1) gene or title STK11(Serine-Threonine kinase11) gene, be positioned human chromosome 19p13.3 position, containing 10 exons, the LKB1 albumen of coding is made up of 433 amino acid, molecular weight is about 50kDa, comprises that kinases region (44-309), N end regulate territory and C end to regulate territory.N end regulates territory containing a nuclear localization sequence, makes LKB1 protein localization in nucleus.A kind of Ser-ine-threonine protein kinase of LKB1 genes encoding, participate in regulate several biological processes, comprise apoptosis, the cell polarity induction etc. of cell-cycle arrest, p53 mediation, the signal paths such as mediation TGF-β, G albumen coupling, in Growth of Cells, differentiation regulation and control, play an important role, in human multiple tissue, all there is expression, maximum with epithelium, testicular spermatogenic tubule, liver, small intestine and skeletal muscle.LKB1 gene is that tumour is easily suffered from syndromes, i.e. the Disease-causing gene of PJ syndrome (Peutz-Jeghers syndrome, PJS) can detect the germline mutation of LKB1 gene in patient PJS up to 90%.The dysfunction of LKB1 gene is also closely related with the generation of the multiple sporadic tumour of whole body, as all there being the inactivation of LKB1 gene in the nonsmall-cell lung cancer of 30-40%, 20% cervical cancer, 19% squamous cell carcinoma and 13% mammary gland wettability duct carcinoma.In addition, LKB1 gene plays an important role in malignant tumour occurs, and studies confirm that LKB1 can make cell cycle arrest at G1 phase, cell growth inhibiting.LKB1 gene is a comparatively general cancer suppressor gene.LKB1 expression conditions is the important middle index of tumour generation and influence prognosis one, the molecular background that discloses LKB1 gene inhibition cell malignant proliferation is had to important meaning, therefore, the detection of LKB1 expression conditions, i.e. the level detection of LKB1 protein level detection or LKB1mRNA is problem demanding prompt solution.
At present, in prior art, the detection of LKB1 expression conditions is mainly the detection of LKB1 albumen, a kind of prior art utilizes rabbit source polyclonal antibody as primary antibodie, then with mark chemochromic reagent or fluorophor two anti-primary antibodie is carried out to mark, after primary antibodie and LKB1 protein binding, judge again the abundance of LKB1 albumen with the color of chemochromic reagent or the fluorescent signal of fluorophor.This indirectly detection method complex steps length consuming time, and because the specificity of polyclonal antibody does not cause by force the generation of non-specific signal, the accuracy of detected result is had a significant impact.Also having a kind of prior art is to utilize the principle of DAB colour developing, to the LKB1 mouse resource monoclonal antibody mark that develops the color, then LKB1 antigen immune groupization dyeing, use again observation by light microscope, there is brown color or tan positive, consider two indexs of ratio that the painted depth of positive cell and positive cell account for observed similar cell, carry out sxemiquantitative judged result.The DAB staining agent using in this method is toxic, for doubtful carcinogenic substance, dangerous, and in addition, chromogenic reagent reaction temperature influence is large, and developing time is restricted, and can not preserve for a long time, and experimenter cannot preserve tissue slice to carry out repeated observation.LKB1mRNA level detection method have not been reported.
Quantum dot has wide excitation spectrum and narrow emmission spectrum (spectral width is between 20-40nm), can in very large range regulate the emmission spectrum of quantum dot by changing the size of quantum dot or the component of change quantum dot kernel.Quantum dot has several outstanding optical characteristics compared with organic fluorescence group: narrow emmission spectrum can reduce spectra overlapping, can distinguish multiple fluorophores simultaneously; Wide excitation spectrum can excite the spectrum of multiple color under single wavelength.In addition quantum dot has light stability and anti-degradation property, little to the toxicity of cell.These unique character make quantum dot can follow the tracks of in vivo multiple cells and the external multiple molecular biosensors of preparation, and because quantum dot has tunable emission wavelength, quantum dot can promote to organize the cell imaging of deep layer.
Summary of the invention
The present invention is intended to solve above-mentioned problems of the prior art, proposes a kind of LKB1mRNA detection method, comprises the following steps:
Preparation LKB1mRNA in situ hybridization probe;
With quantum dot label L KB1mRNA in situ hybridization probe, obtain quantum dot LKB1mRNA in situ hybridization probe;
Carry out in situ hybridization with quantum dot LKB1 mRNA in situ hybridization probe and sample;
Sample after hybridization is placed in to micro-Microscopic observation,
Wherein, described LKB1mrNA in situ hybridization probe be one section with single stranded DNA or the single stranded RNA of LKB1mRNA specific binding; Described sample is cell specimen or tissue sample, and described cell specimen is for organizing printingout, cell climbing sheet or cell smear, and described tissue sample is paraffin section or frozen section.
Preferably, describedly comprise the following steps with quantum dot label L KB1mRNA in situ hybridization probe:
Quantum dot surface is modified with COOH and OH, obtained OH/COOH-quantum dot;
LKB1mRNA in situ hybridization probe, with amido modified, is obtained to amino-LKB1mRNA in situ hybridization probe;
OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe reaction, obtain quantum dot LKB1mRNA in situ hybridization probe.
Preferably, the described step that quantum dot surface is modified with COOH and OH is:
In the mixed solution of DHLA and mercaptoethanol, add quantum dot to react, obtain the first reaction solution;
In the first reaction solution, add chloroform, more centrifugal, collecting precipitation;
By gained precipitation water dissolution, obtain second liquid;
Second liquid is filtered, obtain OH/COOH-quantum dot.
Preferably, described OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe reaction step are:
EDC and sulfo-NHS are dissolved in to phosphoric acid buffer, then add OH/COOH-quantum dot, reaction, obtains the 3rd reaction solution;
In the 3rd reaction solution, add amino-LKB1mRNA in situ hybridization probe, reaction, obtains the 4th reaction solution;
The 4th reaction solution is carried out to chromatography, obtain quantum dot LKB1mRNA in situ hybridization probe.
Preferably, described LKB1mRNA in situ hybridization probe is the specific oligonucleotide sequences of LKB1 gene cDNA, LKB1 gene cRNA or target LKB1mRNA.
The present invention also provides a kind of LKB1mRNA detection kit, comprises quantum dot LKB1mRNA in situ hybridization probe.
Preferably, described test kit also comprises damping fluid, punching liquid, Digestive system, washings and prehybridization working fluid.
Preferably, described washings comprises the first washings, the second washings and the 3rd washings.
Preferably, described test kit also comprises glycerine.
The present invention separately provides a kind of LKB1mrNA to detect gene chip, forms by quantum dot LKB1mRNA in situ hybridization probe being fixed on to surface of solid phase carriers.
Preferably, described solid phase carrier is selected from nitrocellulose filter, nylon membrane, polystyrene, sheet glass, silicon chip, particulate or plastic sheet.
LKB1mRNA detection method of the present invention utilizes quantum dot to modify specific single-chain DNA or the RNA of target LKB1mRNA, form quantum dot LKB1mRNA in situ hybridization probe, utilize quantum dot LKB1mRNA in situ hybridization probe to be combined with LKB1mRNA, by quantum dot-labeled upper to LKB1mRNA, utilize fluorescence that quantum dot sends to LKB1mRNA position, qualitative and quantitative examination.Cell sample or tissue samples are made into after sample, add quantum dot LKB1mRNA in situ hybridization probe to carry out in situ hybridization, and the sample after hybridization is placed in to fluorescence microscopy Microscopic observation.After the combination of LKB1mRNA and quantum dot LKB1mRNA in situ hybridization probe specificity, LKB1mRNA sends fluorescence, can send the positive cell of cell of fluorescence, can not send the negative cell of fluorescence, also can account for by considering positive cell fluorescence intensity and positive cell two indexs of ratio of observed similar cell, carry out sxemiquantitative judged result.
Beneficial effect of the present invention is, detection method of the present invention is used quantum dot LKB1mRNA in situ hybridization probe, and quantum dot is less than other fluorescence dye toxicity, uses saferly, and painted sample can be preserved for a long time, carries out repeated observation; The beneficial effect of detection method of the present invention is also simple to operate, highly sensitive.
Brief description of the drawings
Fig. 1 is normal breast cell smear fluorescence in situ hybridization result figure under fluorescent microscope.
Fig. 2 is normal gastric cell smear fluorescence in situ hybridization result figure under fluorescent microscope.
Embodiment
In order to make those skilled in the art better understand the application's technical scheme, below in conjunction with the accompanying drawing in the embodiment of the present application, the technical scheme in the embodiment of the present application is carried out to clear, complete description.
LKB1 method of protein detection of the present invention realizes by the following technical solutions:
Preparation LKB1mRNA in situ hybridization probe;
With quantum dot label L KB1mRNA in situ hybridization probe, obtain quantum dot LKB1mRNA in situ hybridization probe;
Carry out in situ hybridization with quantum dot LKB1mRNA in situ hybridization probe and sample;
Reacted sample is placed in to micro-Microscopic observation,
Wherein, described LKB1mRNA in situ hybridization probe be one section with single stranded DNA or the single stranded RNA sequence of LKB1mRNA specific binding; Described sample is cell specimen or tissue sample, and described cell specimen is for organizing printingout, cell climbing sheet or cell smear, and described tissue sample is paraffin section or frozen section.
LKB1mRNA in situ hybridization probe can be selected the specific oligonucleotide sequences of LKB1 gene cDNA, LKB1 gene cRNA or target LKB1mRNA.
Quantum dot can be selected CdSe, CdTe, CdSe/ZnS, CdTe/ZnS, CdTe/CdS/ZnS, CdSe/CdS/ZnS, CdSe/CdZnS, CdSe:Mn 2+, CdTe:Mn 2+, CdSe:Zn 2+or CdTe:Zn 2+, be not limited to above.The quantum dot using in the present embodiment is CdSe/ZnS.
In the present embodiment, the preparation of quantum dot LKB1mRNA in situ hybridization probe comprises the following steps:
Quantum dot surface is modified with COOH and OH, obtained OH/COOH-quantum dot;
LKB1mRNA in situ hybridization probe, with amido modified, is obtained to amino-LKB1mRNA in situ hybridization probe;
OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe reaction, obtain quantum dot LKB1mRNA in situ hybridization probe.
Preferably, the described step that quantum dot surface is modified with COOH and OH is:
DHLA(Dihydrolipoic Acid, Thioctic acid, dihydro-) molecule one end is sulfydryl, the other end is carboxyl (COOH); One end of mercaptoethanol molecule is sulfydryl, and the other end is hydroxyl (OH), and DHLA is combined by golden sulfide linkage with quantum dot with the sulfydryl of mercaptoethanol, forms surface by COOH and the common coated OH/COOH-quantum dot of OH.
OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe, by amino crosslinked and the amino and crosslinked combination of OH with COOH, form quantum dot LKB1mRNA in situ hybridization probe.
The crosslinked of amino realized in the following manner, EDC(1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide), sulfo-NHS(N-Hydroxysulfosuccinimide, N-hydroxy-succinamide) mix with OH/COOH-quantum dot, OH/COOH-quantum dot and sulfo-NHS form active intermediate OH/COOH-quantum dot-NHS under EDC catalysis, add again amino-LKB1mRNA in situ hybridization probe, the N-terminal of amino-LKB1mRNA in situ hybridization probe and COOH and the OH ligation of OH/COOH-quantum dot, obtain quantum dot LKB1mRNA in situ hybridization probe.
In addition, quantum dot LKB1mRNA in situ hybridization probe and sample carry out in situ hybridization and comprise the following steps:
Damping fluid is processed sample, makes histocyte recover water-based;
With processing sample in the liquid of hole, change histiocytic permeability, wash with washings;
Digestive system is processed sample, with the first washings and the flushing of the second washings;
Prehybridization processing: prehybridization working fluid is processed sample, covers in situ hybridization special cap slide, prevents that prehybridization solution from killing;
Prehybridization is processed rear with the second washings flushing;
Quantum dot LKB1mRNA in situ hybridization probe hybridization working fluid mixes, and the processing of quantum dot LKB1mRNA in situ hybridization probe deformations, obtains mixed solution;
Above-mentioned gained mixed solution is processed to sample, with the second washings and the flushing of the 3rd washings;
Use glycerine mounting in fluorescence microscopy Microscopic observation results of hybridization sample.
Wherein, described damping fluid is preferably citrate buffer; The first described washings is preferably PBS solution; The second described washings is preferably SSC solution; The 3rd described washings is preferably TBS solution; The formula optimization of described prehybridization working fluid is: 4 × SSC, 50% methane amide, 100ng/ μ l BSA, 1 × Dehardt solution, 0.3%Triton X-100,0.5
MMribonucleosidevanadylcomplexes and1ng/ml salmon sperm dna.
The present invention also provides LKB1mRNA detection kit, comprise quantum dot LKB1mRNA in situ hybridization probe, also comprise damping fluid, punching liquid, Digestive system, washings and prehybridization working fluid and glycerine, described washings comprises the first washings, the second washings and the 3rd washings.
The using method of LKB1mRNA detection kit of the present invention is as follows:
Sample is soaked with damping fluid;
With punching liquid processing sample;
Process sample with Digestive system;
With prehybridization working fluid processing sample;
Quantum dot LKB1mRNA in situ hybridization probe and sample carry out in situ hybridization;
Micro-Microscopic observation after glycerine mounting.
The present invention separately provides a kind of LKB1mRNA to detect gene chip; form by quantum dot LKB1mRNA in situ hybridization probe being fixed on to surface of solid phase carriers, described solid phase carrier is selected from nitrocellulose filter, nylon membrane, polystyrene, sheet glass, silicon chip, particulate or plastic sheet.
The using method that LKB1mRNA of the present invention detects gene chip is as follows:
The total RNA of testing sample extracts;
Total RNA reverse transcription generates total cDNA;
The amplification of LKB1cDNA;
The mark of LKB1cDNA;
Quantum dot LKB1mRNA in situ hybridization probe on LKB1cDNA through mark and gene chip is hybridized.
Be below embodiment.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Embodiment 1
In the present embodiment, select cDNA the first chain of LKB1 as LKB1mRNA in situ hybridization probe, the cDNA sequence of LKB1 is as shown in SEQ ID No.1.
LKB1mRNA detecting step:
(1) OH/COOH of quantum dot modifies
A.0.6ml DHLA and 0.9ml mercaptoethanol and 1.4ml methyl alcohol mix, and add subsequently 130mgCdSe/ZnS quantum dot, and 65 DEG C of stirrings are spent the night, and obtain OH/COOH-quantum dot solution; Add 3ml chloroform to make its precipitation; The centrifugal 5min collecting precipitation of 14000rpm;
B. the precipitation of collecting in step a is dissolved in to 1ml methyl alcohol, then with 3ml chloroform precipitation, the centrifugal 5min of 14000rpm collecting precipitation again;
C. gained precipitation in step b is dissolved in to 1.6ml water, with 0.2 μ m membrane filtration, obtains OH/COOH-quantum dot;
(2) OH/COOH-quantum dot and LKB1mRNA in situ hybridization probe covalent attachment
A. 2.5mg EDC, 1.25mgSulfo-NHS are dissolved in 60 μ l phosphoric acid buffers;
B. dissolve OH/COOH-quantum dot with gained solution in step a, OH/COOH-quantum dot concentration is 3 μ mol/L, and room temperature is placed 15min;
C.60 in μ l50 μ M amino-LKB1-cDNA in situ hybridization probe and step b, gained solution mixes, and at 30 DEG C, reacts 30min;
D. remove excessive EDC and Sulfo-NHS with NAP5 column chromatography, obtain quantum dot LKB1mRNA in situ hybridization probe;
(3) quantum dot LKB1mRNA in situ hybridization probe and sample to be measured carry out in situ hybridization
A. use 0.1mol/L citrate buffer immersed specimen, under room temperature, place 10min;
B. with punching liquid immersed specimen, under room temperature, place 10min, then use 1 × PBS solution to rinse three times;
C. process sample with 0.5 μ g/ml Proteinase K, at 42 DEG C, place 20min, then use 1 × PBS solution to rinse three times, rear use 0.2 × S SC solution rinses 3min under room temperature;
D. process sample with prehybridization working fluid, hatch 4-8h in 42 DEG C of wet boxes, between incubation period, cover sample with in situ hybridization special cap slide;
E. after hatching, under room temperature, rinse sample three times with 0.2 × SSC solution, each flush time 5min;
F. quantum dot LKB1mRNA in situ hybridization probe is dissolved in prehybridization working fluid and is mixed and heated to 70 DEG C of sex change 15min, and the concentration of quantum dot LKB1mRNA in situ hybridization probe is 1nmol/L;
G. cover sample with step f gained solution and hatch 8-12h in 42 DEG C of wet boxes, between incubation period, cover sample with in situ hybridization special cap slide;
H. after hatching, with 2 × SSC solution, rinse samples three times in 37 DEG C, each flush time 5min, repeats above-mentioned rinse step once, then rinses 3-5 time flush time 5min at every turn in 37 DEG C with 0.1mol/L TBS solution;
I. glycerine mounting is in fluorescence microscopy Microscopic observation results of hybridization.
Wherein, the building-up process of CdSe/ZnS quantum dot is: after 30mg CdO and 600mg lauric acid (dodecanoicacid) are mixed, put into three-necked flask together with 4g99%TOPO and 4g90%HDA, be filled with argon gas, be heated to 300 DEG C of constant temperature and dissolve to CdO; 180mg Se is dissolved in to 2ml TOP, injects fast reaction flask, then add 10ml toluene termination reaction; Be annealed to 150 DEG C of constant temperature 30min; Products therefrom is cooling and make CdSe quantum dot precipitation with anhydrous methanol, gained is deposited in to 7g90%TOPO resuspended, obtain CdSe quantum dot solution; Gained CdSe quantum dot solution is heated to 80 DEG C, then adds 2.5mlZnS, be filled with argon gas, continue to be heated to 180 DEG C of constant temperature 30min, make CdSe quantum dot form ZnS shell outward, after annealing to 120 DEG C constant temperature 2.5h, obtains CdSe/Zns quantum dot; Make CdSe/Zns quantum dot precipitation with methyl alcohol, collecting precipitation, and be stored in chloroform for subsequent use.
Prehybridization Working solution prescription: 4 × SSC solution, 50% methane amide, 100ng/ μ l BSA solution, 1 × Dehardt solution, 0.3%Triton X-100(Triton X-100), 0.5mmol/Lribonucleosidevanadylcomplexes(ribonucleoside vanadyl complexes) and 1ng/ml salmon sperm dna.
The preparation of LKB1mRNA probe: carry out the synthetic LKB1cDNA of acquisition of DNA the first chain according to SEQ ID No.1, using this as LKB1mrNA probe.
Application examples 1
Normal breast cell is made to cell smear, normal mammary gland cell smear is observed by the LKB1mRNA detection method in embodiment 1.Fig. 1 is observe and normal breast cell quantum dot-LKB1mRNA Probe In Situ Hybridization under fluorescent microscope.
Application examples 2
Normal gastric cell is made to cell smear, normal gastric cells smear is observed by the LKB1mRNA detection method in embodiment 1.Fig. 2 be under fluorescent microscope observe with quantum dot-LKB1mRNA Probe In Situ Hybridization normal gastric cell.
embodiment 2
Make LKB1mRNA detection kit according to following formula:
Quantum dot LKB1mRNA in situ hybridization probe: according to the synthetic quantum dot LKB1mRNA in situ hybridization probe in embodiment 1 step (1)-(3);
Damping fluid: 0.1mol/L citrate buffer;
Punching liquid;
Digestive system: 0.5 μ g/ml Proteinase K;
Washings: described washings comprises the first washings, the second washings and the 3rd washings,
Prehybridization working fluid: 4 × SSC solution, 50% methane amide, 100ng/ μ l BSA solution, 1 × Dehardt solution, 0.3%Triton X-100(Triton X-100), 0.5mmol/Lribonucleosidevanadylcomplexes(ribonucleoside vanadyl complexes) and 1ng/ml salmon sperm dna;
Glycerine.
The using method of LKB1mRNA detection kit is according to embodiment 1 step (3).
embodiment 3
Make LKB1mRNA according to following formula and detect gene chip:
Quantum dot LKB1mRNA in situ hybridization probe preparation: according to the synthetic quantum dot LKB1mRNA in situ hybridization probe in embodiment 1 step (1)-(3);
Use 0.5mol/L NaHCO 3solution dilution quantum dot LKB1mRNA in situ hybridization probe, joins the probe after dilution in microwell plate, adds isopyknic sampling liquid, fully mixes;
BiodyneC nylon membrane is cut to suitable size;
Under room temperature, hatch 5min with 16%w/v EDTA and BiodyneC nylon membrane, rear by rinsed with deionized water, and blot with filter paper;
Get probe solution point sample on the BiodyneC nylon membrane preparing with point sample instrument, under room temperature, hatch 10min;
Absorb BiodyneC nylon membrane surface liquid, BiodyneC nylon membrane is put into 0.1mol/L NaOH and soak 10min, rear with 2*SSPE rinsing 5min, then clean with EDTA solution;
BiodyneC nylon membrane is put into clean plastics bag, and add EDTA, sealing is preserved, for subsequent use.
The BiodyneC nylon membrane that is fixed with quantum dot LKB1mRNA in situ hybridization probe of above-mentioned preparation is LKB1mRNA and detects gene chip.
Utilize the LKB1mRNA of the present embodiment to detect LKB1mRNA in genechip detection stomach cancer cell BGC823, step is as follows:
The total RNA of stomach cancer cell BGC823 extracts;
Total RNA reverse transcription generates total cDNA;
The double-stranded preparation of LKB1cDNA: according to the cDNA sequence of people LKB1 gene in GenBank, with Oligo5.0 software assistant analysis, design primer SEQ ID No.2 and SEQ ID No.3, increase taking total cDNA as masterplate, PCR reaction conditions is 94 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C are extended 1min, more than carry out 35 circulations, last 72 DEG C are extended 10min, and after gained PCR product is purified, gained purified product is LKB1cDNA two strands;
The double-stranded mark of LKB1cDNA: LKB1cDNA is carried out to mark with bipyridyl ruthenium, obtain bipyridyl ruthenium-LKB1cDNA;
Bipyridyl ruthenium-LKB1cDNA and LKB1mRNA are detected to gene chip hybridization.
When bipyridyl ruthenium exists, shift with quantum dot generation Photoinduced Electron, quantum dot fluorescence is suppressed, can't detect any signal, bipyridyl ruthenium-LKB1cDNA content is higher, and repressed quantum dot fluorescence signal is more, and the amount of bipyridyl ruthenium-LKB1cDNA is directly proportional to LKB1mRNA content, be that LKB1-mRNA content is higher, quantum dot fluorescence is suppressed more, and signal is more weak.
Although the present invention is described with reference to current preferred embodiments; but those skilled in the art will be understood that; above-mentioned preferred embodiments is only used for illustrating the present invention; not be used for limiting protection scope of the present invention; any within the spirit and principles in the present invention scope; any modification of doing, equivalent replacement, improvement etc., within all should being included in the scope of the present invention.
Figure IDA00002629820700011
Figure IDA00002629820700031

Claims (10)

1. a LKB1mRNA detection method, is characterized in that, comprises the following steps:
Preparation LKB1mRNA in situ hybridization probe;
With quantum dot label L KB1mRNA in situ hybridization probe, obtain quantum dot LKB1mRNA in situ hybridization probe;
Carry out in situ hybridization with quantum dot LKB1mRNA in situ hybridization probe and sample;
Sample after hybridization is placed in to micro-Microscopic observation,
Wherein, described LKB1mRNA in situ hybridization probe be one section with single stranded DNA or the single stranded RNA of LKB1mRNA specific binding; Described sample is cell specimen or tissue sample, and described cell specimen is for organizing printingout, cell climbing sheet or cell smear, and described tissue sample is paraffin section or frozen section.
2. detection method according to claim 1, is characterized in that, described comprises the following steps with quantum dot label L KB1mRNA in situ hybridization probe:
Quantum dot surface is modified with COOH and OH, obtained OH/COOH-quantum dot;
LKB1mRNA in situ hybridization probe, with amido modified, is obtained to amino-LKB1mRNA in situ hybridization probe;
OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe reaction, obtain quantum dot LKB1mRNA in situ hybridization probe.
3. detection method according to claim 2, is characterized in that, the described step that quantum dot surface is modified with COOH and OH is:
In the mixed solution of DHLA and mercaptoethanol, add quantum dot to react, obtain the first reaction solution;
In the first reaction solution, add chloroform, more centrifugal, collecting precipitation;
By gained precipitation water dissolution, obtain second liquid;
Second liquid is filtered, obtain OH/COOH-quantum dot.
4. detection method according to claim 2, is characterized in that, described OH/COOH-quantum dot and amino-LKB1mRNA in situ hybridization probe reaction step are:
EDC and sulfo-NHS are dissolved in to phosphoric acid buffer, then add OH/COOH-quantum dot, reaction, obtains the 3rd reaction solution;
In the 3rd reaction solution, add amino-LKB1mRNA in situ hybridization probe, reaction, obtains the 4th reaction solution;
The 4th reaction solution is carried out to chromatography, obtain quantum dot LKB1mRNA in situ hybridization probe.
5. according to the detection method described in claim 1-4 any one, it is characterized in that, described LKB1mRNA in situ hybridization probe is the specific oligonucleotide sequences of LKB1 gene cDNA, LKB1 gene cRNA or target LKB1mRNA.
6. a LKB1mRNA detection kit, is characterized in that, comprises quantum dot LKB1mRNA in situ hybridization probe.
7. test kit according to claim 6, is characterized in that, also comprises damping fluid, punching liquid, Digestive system, washings and prehybridization working fluid.
8. test kit according to claim 7, is characterized in that, described washings comprises the first washings, the second washings and the 3rd washings.
9. LKB1mRNA detects a gene chip, it is characterized in that, forms by quantum dot LKB1mRNA in situ hybridization probe being fixed on to surface of solid phase carriers.
10. gene chip according to claim 9, is characterized in that, described solid phase carrier is selected from nitrocellulose filter, nylon membrane, polystyrene, sheet glass, silicon chip, particulate or plastic sheet.
CN201210562544.8A 2012-12-21 2012-12-21 LKB1(Liver Kinase B1)mRNA (messenger RNA) detection method, kit and gene chip Pending CN103882099A (en)

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EP3198037A4 (en) * 2014-09-22 2018-03-21 The Regents of the University of California Single molecule rna detection
CN108949913A (en) * 2018-07-31 2018-12-07 四川大学华西医院 Gene detecting kit and its application comprising universal fluorescent abatement probe nucleic acid molecule

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDREW ROWAN ET AL.: "In situ analysis of LKB1/STK11 mRNA expression in human normal tissues and tumours", 《THE JOURNAL OF PATHOLOGY》 *
黄萍等: "量子点荧光探针在生物医学研究中的应用进展", 《世界华人消化杂志》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3198037A4 (en) * 2014-09-22 2018-03-21 The Regents of the University of California Single molecule rna detection
CN108949913A (en) * 2018-07-31 2018-12-07 四川大学华西医院 Gene detecting kit and its application comprising universal fluorescent abatement probe nucleic acid molecule
CN108949913B (en) * 2018-07-31 2021-05-25 四川大学华西医院 Gene detection kit containing universal fluorescent reduction probe nucleic acid molecules and application thereof

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Application publication date: 20140625