CN101240345A - In-situ combination detection method for miRNA expression and cell apoptosis - Google Patents
In-situ combination detection method for miRNA expression and cell apoptosis Download PDFInfo
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- CN101240345A CN101240345A CNA2008100523687A CN200810052368A CN101240345A CN 101240345 A CN101240345 A CN 101240345A CN A2008100523687 A CNA2008100523687 A CN A2008100523687A CN 200810052368 A CN200810052368 A CN 200810052368A CN 101240345 A CN101240345 A CN 101240345A
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Abstract
The invention discloses a method for in-situ joint detection of the expression of miRNA and fluorescence in-situ hybridization detection of apoptosis. The method comprises: synthesizing miRNA specific probe modified by LNA, labeling the probe with digoxin, purifying, biotinzing mouse anti-digoxin and constructing affinity-fluorescence labeling system and other basic steps. The invention can in-situ joint detect the expression of miRNA and apoptosis at the same time. The detection method of the invention converts the expression condition of miRNA to visible fluorescence signal, getting a good in-situ detection effect and filling a gap in the application of miRNA in-suit fluorescence detection.
Description
[technical field]
The present invention relates to the improvement of miRNA in-situ detection method.Particularly a kind of in-situ combination detects expression and the apoptotic fluorescent in situ hybridization detecting method of miRNA.
[background technology]
Recent study is found, the microRNA that one class is new (microRNA or miRNA) is meant some special small-sized non-coding RNA of the about 21～25nt of length, these miRNA can discern specific target mRNA, and combine with its 3 ' non-translational region by complete or incomplete complementary mode, realize post-transcriptional level by promoting said target mrna degraded and/or suppress translation process and bring into play the effect that negative regulator gene is expressed.Human by inference nearly 30% genetic expression is subjected to the accurate adjusting of miRNA.The miRNA that has determined has kind more than 300 at present, and according to the supposition of miRNA database Sanger, its total amount may surpass 1000, occupies 3% of people's gene group.Because miRNA extensively exists in vivo and be very accurate to its downstream target gene regulation and control, therefore, the research of miRNA has become new focus in the fields such as tumour, growth, stem cell.
The MiRNA mean length has only 22bp, detects relatively difficulty by ordinary method.Kai Fa miRNA real-time PCR and Northern blot have solved the augmentation detection about miRNA in recent years; But the Subcellular Localization of miRNA, and on research such as the mechanism of miRNA and the said target mrna effect level, above-mentioned experimental technique the information that can provide just seem very limited.Simultaneously expression and the apoptotic relation of part miRNA are very close, so in-situ combination detects the expression of little RNA and apoptotic dose of box and detection method thereof and needs foundation.
[summary of the invention]
The objective of the invention is in order to overcome the deficiencies in the prior art, and provide a kind of in-situ combination to detect miRNA and apoptotic detection method, this method does not have radiation, pollution-free, easy to use.
The scheme that the present invention is adopted for achieving the above object discloses expression and the apoptosis method that a kind of in-situ combination detects little RNA, it is characterized in that described method comprises: will carry out locked nucleic acid according to the rna probe of the ripe body base sequence design of miRNA and modify (LNA, locked nuclear acid modification), make the miRNA specific probe that LNA modifies; In specific probe 3 ' end mark digoxin, obtain the miRNA specific probe liquid that LNA modifies; Amplify the miRNA expression signal by immune response; Realize the expression of in situ detection miRNA at last by fluorescently-labeled avidin (Avidin).
After described miRNA expressed in situ detects, detect the original position apoptosis by the TUNEL method.Thereby expression and apoptosis that can synchronous detection miRNA.
The invention has the beneficial effects as follows: the present invention is converted into the visible fluorescence signal with the expressed in situ situation of miRNA; The expression and the apoptosis situation that simultaneously can in-situ combination detect miRNA; Reach directly perceived, good in situ detection effect, filled up the application blank in miRNA original position fluoroscopic examination field.
[description of drawings]
Fig. 1 is viable cell miRNA in situ hybridization result;
Fig. 2 is paraffin section miRNA in situ hybridization result;
Fig. 3 is that MiR-21 in situ hybridization and TUNEL method detect apoptosis.
Be described in detail with reference to accompanying drawing below in conjunction with embodiments of the invention.
[embodiment]
The following stated; only for the preferable embodiment of the present invention, but protection scope of the present invention is not limited thereto, and anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Technical scheme of the present invention is summarized as follows:
One, the preparation of LNA-miRNA probe
1. the miRNA antisense RNA probes of the required detection of chemosynthesis;
2. above-mentioned synthetic probe and absolute excessive digoxin are spent the night in liquid phase environment hybridization, by chromatography, the dialysis means are removed excessive digoxin and are not participated in the rna probe of reaction.Gained solution is labeled as hybridization probe liquid.
Two, the preparation of in situ hybridization reactive system
1. to make 10 * stock solution standby for stomach en-, solution A;
2.40% deionized formamide, 10% T 500,1 * Denhardt liquid, 4 * SSC, 1g/L tRNA, 1g/L thermally denature salmon sperm dna, mixed solution gets solution B;
3. to be mixed with concentration be 20% solution to the lowlenthal serum albumin, obtains solution C;
4. with after the vitamin H activation,, get solution D with mouse-anti digoxin bonding mark;
5. with after the avidin activation,, get solution E with Indodicarbocyanine (Cy3) bonding mark.
Three, the preparation of required other the reagent/damping fluids of in situ hybridization:
1. in situ hybridization is with phosphate buffered saline buffer (PBS):
A liquid: Na
2HPO
428.4g, Na
2HPO
4H
2O 31.61g, Na
2HPO
42H
2O 35.6g, Na
2HPO
47H
2O 53.63g, Na
2HPO
412H
2O 71.64g adds distilled water to 1000ml;
B liquid: NaH
2PO
424.0g, NaH
2PO
4H
2O 27.6g, NaH
2PO
42H
2O 31.21g adds distilled water to 1000ml;
According to 81: 19 ratios get corresponding volume A respectively, B liquid mixes, and adds and is equivalent to mixed solution volumetrical distilled water, mix.Add diethylpyrocarbonate (DEPC) according to 1 ‰ concentration, it is even fully to be mixed, and standing over night is standby;
2.DEPC processing distilled water:
Distilled water adds diethylpyrocarbonate (DEPC) according to 1 ‰ concentration, and it is even fully to be mixed, and standing over night is standby;
3. stationary liquid:
4% Paraformaldehyde 96 (PFA)/in situ hybridization PBS solution: 4g Paraformaldehyde 96 powder is dissolved in the 0.1M PBS solution makes;
4. 1%PFA/0.1M PBS solution:
1g Paraformaldehyde 96 powder is dissolved in the 0.1M PBS solution makes;
5. 3% citric acid solution:
The 0.3g citric acid is dissolved in 10ml DEPC treating water, and thorough mixing is evenly standby;
6. 0.1%Tritoon-100/ in situ hybridization PBS solution
1ml Tritoon-100 is added 100ml in situ hybridization PBS solution, mix standby;
7.SSC buffered soln:
2 * SSC: get NaCl 17.53g; Trisodium Citrate 8.82g; Add water to 1000ml, transfer PH to 7.0 with 10mol/L NaOH;
0.5 * SSC: get 2 * SSC solution 50ml, add DEPC and handle distilled water to 200ml;
0.2 * SSC: get 2 * SSC solution 20ml, add DEPC and handle distilled water to 200ml.
Four, apoptosis in situ detection
TUNEL apoptosis detection kit (Roche company, Switzerland);
Washings: 0.01M PBS (pH 7.4);
Confining liquid: 3%H
2O
2/ methyl alcohol;
Stationary liquid: 4% Paraformaldehyde 96 is dissolved in PBS (pH 7.4), fresh preparation;
Saturating film liquid: 0.1%Triton X-100 is dissolved in 0.1% Trisodium Citrate, fresh preparation;
The TUNEL marking fluid: afterwards ice bath is stand-by according to mixing at 1: 9 with 2 pipes for 1 pipe.
The concrete example 1 that detects:
Detection reagent and set up process
One, the preparation of hybridization probe:
1. reagent
Chemosynthesis sense-rna oligonucleotide (the lucky agate product in Shanghai); Digoxin (Sigma product);
2. instrument
Hybrid heater (Bio-Rad product); Chromatography column (Millipore product).
3. method
The chemosynthesis oligonucleotide probe spends the night in liquid phase environment hybridization with absolute excessive digoxin, and by chromatography, the dialysis means are removed excessive digoxin and do not participated in the rna probe of reaction.Gained solution is labeled as hybridization probe liquid.
Two, miRNA in situ hybridization reaction system preparation:
1. reagent
Stomach en-, deionized formamide, T 500, Denhardt liquid, lowlenthal serum albumin, vitamin H, avidin, mouse-anti DigiTAb, Indodicarbocyanine (Sigma product); TRNA, thermally denature salmon sperm dna (SMDNA product).
2. instrument
Hybrid heater (Bio-Rad product).
3. method
1) 40% deionized formamide, 10% T 500,1 * Denhardt liquid, 4 * SSC, 1g/L tRNA, 1g/L thermally denature salmon sperm dna mixes hybridization and makes solution B;
2) to be mixed with concentration be 20% solution to the lowlenthal serum albumin, obtains solution C;
3),, get solution D with mouse-anti digoxin bonding mark with after the vitamin H activation;
4),, get solution E with Indodicarbocyanine (Cy3) bonding mark with after the avidin activation.
Three: viable cell miRNA in situ detection
1. conventional cell cultures, creep plate preparation;
2. cell is fixing: 4%PFA/ in situ hybridization fixedly 30min of PBS solution room temperature, and distilled water is fully washed;
3.30% hydrogen peroxide adds 50 parts of methanol mixed for 1 part, room temperature treatment 30min, and deactivating endogenous peroxydase, distilled water is fully washed;
The concentrated type stomach en-(1ml 3% citric acid adds 2 solution A, mixing) of 4.3% citric acid dilution, 37 ℃ or incubated at room 10min.Originally be that hybridization is washed 3 * 5min with PBS solution, DEPC handles distilled water and washes once;
5.37 the 0.1%Triton X-100/ in situ hybridization of ℃ preheating is hatched 30min with PBS solution, in situ hybridization PBS thorough washing;
6.1%PFA/ in situ hybridization fixedly 10min of PBS solution room temperature, DEPC handles distilled water washing 3 times;
7. every cell adds 40 μ l solution B, hatches 4 hours for 38 ℃, siphons away unnecessary liquid, does not wash;
8. every section adds 40 μ l hybridization probe liquid, is added on the cell, and 38 ℃ of hybridization are spent the night;
9.37 2 * SSC solution washing, the 2 * 10min of ℃ preheating, 0.5 * SSC solution washing, the 1 * 15min of 37 ℃ of preheatings, 0.2 * SSC solution washing, the 1 * 15min of 37 ℃ of preheatings;
10. dropping solution C is hatched 30min for 37 ℃, gets rid of unnecessary liquid, does not wash;
11. the dropping solution D is hatched 60min for 37 ℃, in situ hybridization is washed 4 * 5min with PBS;
12. the dropping solution E, 37 ℃ of lucifuges were hatched 2 hours, in situ hybridization PBS thorough washing;
13.Hochest33258 solution is redyed nucleus, in situ hybridization PBS thorough washing;
14. anti-quencher mounting is prepared microscopy;
15.FV-1000 laser confocal microscope is observed.
The detected result picture is seen shown in the accompanying drawing 1.Be distributed in to the disperse of the positive hybridization fluorescent signal of caption Cy3 U251 cell karyon, visual representation the Subcellular Localization situation of little RNA on cell climbing sheet.
Four, apoptosis in situ detection
TUNEL apoptosis detection kit (Roche company, Switzerland)
Washings: 0.01M PBS (pH 7.4).
Confining liquid: 3%H2O2/ methyl alcohol.
Stationary liquid: 4% Paraformaldehyde 96 is dissolved in PBS (pH 7.4), fresh preparation.
Saturating film liquid: 0.1%Triton X-100 is dissolved in 0.1% Trisodium Citrate, fresh preparation.
The TUNEL marking fluid: afterwards ice bath is stand-by according to mixing at 1: 9 with 2 pipes for 1 pipe.
The apogen position detecting method is to connect above-mentioned step 3, viable cell miRNA in situ detection step 12:
Slide dipping bath 5 minutes * 2 times in 0.01M PBS (pH7.4) damping fluid;
The liquid 20-25 μ l that labels is hatched after 60 minutes 4 ℃ and is spent the night for 37 ℃ in the wet box;
PBS (pH7.4) jog is washed 5 minutes * 3 times, room temperature;
Hochest33258 solution is redyed nucleus, in situ hybridization PBS thorough washing; Anti-quencher mounting; The FV-1000 laser confocal scanning microscope is observed.
The concrete example 2 that detects
One, paraffin section miRNA in situ detection
1. poly-lysine is handled slide glass;
2. routine paraffin wax section preparation; 6～8 μ l;
3. the paraffin section routine dewaxes to water;
4.30% hydrogen peroxide adds 10 parts of distilled water for 1 part and mixes room temperature treatment 10min, deactivating endogenous peroxydase;
5. expose the fragment of probe specificity: the concentrated type stomach en-(the 1ml3% citric acid adds 2 solution A, mixing) of 3% citric acid dilution, 37 ℃ or incubated at room 3～30min in conjunction with miRNA.Originally be that hybridization is washed 3 * 5min with PBS, DEPC handles distilled water and washes once;
6.37 0.1% Triton X-100/ in situ hybridization of ℃ preheating is hatched 30min with PBS solution, in situ hybridization PBS thorough washing;
7.1%PFA/ in situ hybridization fixedly 10min of PBS solution room temperature, DEPC handles distilled water washing 3 times;
8. prehybridization: every section adds 40 μ l solution B, is added in the section, hatches 4 hours for 42 ℃, siphons away unnecessary liquid, does not wash;
9. every section adds 40 μ l hybridization probe liquid, is added in the section, and 42 ℃ of hybridization are spent the night;
10.37 2 * SSC solution washing, the 2 * 5min of ℃ preheating, 0.5 * SSC solution washing, the 1 * 15min of 37 ℃ of preheatings, 0.2 * SSC solution washing, the 1 * 15min of 37 ℃ of preheatings;
11. the dropping solution C, 37 ℃ of sealing 30min get rid of unnecessary liquid, do not wash;
12. the dropping solution D, 37 ℃ of sealing 60min, in situ hybridization is washed 4 * 5min with PBS;
13. the dropping solution E, 37 ℃ of lucifuges were hatched 2 hours, in situ hybridization PBS thorough washing;
14.Hochest33258 solution is redyed nucleus, in situ hybridization PBS thorough washing;
15. anti-quencher mounting is prepared microscopy;
16.FV-1000 laser confocal microscope is observed.
The detected result picture is seen accompanying drawing 2.Be distributed in to the disperse of the positive hybridization fluorescent signal of caption Cy3 the tumour cell endochylema.
Two, apoptosis in situ detection
Connect above-mentioned step 1, paraffin section miRNA in situ detection step 13 back:
Slide dipping bath 5 minutes * 2 times in 0.01M PBS (pH7.4) damping fluid;
The liquid 25 μ l that label are hatched after 60 minutes 4 ℃ and are spent the night for 37 ℃ in the wet box,
M PBS (pH7.4) jog is washed 5 minutes * 3 times, room temperature;
Hochest33258 solution is redyed nucleus, in situ hybridization PBS thorough washing; Anti-quencher mounting; The FV-1000 laser confocal scanning microscope is observed.
The detected result picture is seen accompanying drawing 3.The expression of the little RNA of caption and apoptosis have direct relation, and the positive signal of the two has the Subcellular Localization performance of notable difference on paraffin section.
Claims (4)
1, a kind of in-situ combination detects the method for the expression of miRNA, it is characterized in that described method comprises: will carry out locked nucleic acid (LNA) according to the rna probe of the ripe body base sequence design of miRNA and modify, and make the miRNA specific probe that LNA modifies; In specific probe 3 ' end mark digoxin, obtain the miRNA specific probe liquid that LNA modifies; Amplify the miRNA expression signal by immune response; Realize the expression of in situ detection miRNA at last by fluorescently-labeled avidin.
2, method according to claim 1, it is characterized in that described miRNA expressed in situ detects after, detect the original position apoptosis by the TUNEL method.
3, method according to claim 1, it is characterized in that comprising that following main ingredient: LNA modifies miRNA specific probe liquid, the miRNA specific probe liquid that biotinylation mouse-anti digoxin and avidin-fluorescent mark system: LNA are modified is by chemosynthesis miRNA antisense rna oligonucleotide, behind the purifying as probe; Described synthetic probe spends the night in liquid phase environment hybridization with excessive digoxin again, and by chromatography, the dialysis means are removed excessive digoxin and do not participated in the rna probe of reaction, and gained solution is labeled as hybridization probe liquid.
4, method according to claim 1 is characterized in that the miRNA in situ hybridization reaction system of described method is:
A) stomach en-is made 10 * stock solution, gets solution A;
B) 40% deionized formamide, 10% T 500,1 * Denhardt liquid, 4 * SSC, 1g/LtRNA, 1g/L thermally denature salmon sperm dna, mixed solution gets solution B;
C) to be mixed with concentration be 20% solution to the lowlenthal serum albumin, obtains solution C;
D),, get solution D with mouse-anti digoxin bonding mark with after the vitamin H activation;
E),, get solution E with Indodicarbocyanine (Cy3) bonding mark with after the avidin activation.
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| CN102382893A (en) * | 2011-11-23 | 2012-03-21 | 博生吉医药科技(苏州)有限公司 | Specificity quantitive detection for cell apoptosis |
| CN102533984A (en) * | 2011-12-19 | 2012-07-04 | 苏州福英基因科技有限公司 | Horizontal in-situ hybridization detection kit and detection method as well as application for MICRORNA17-3P in earlier stage of cancer pathologic evolution |
| CN102732619A (en) * | 2012-06-06 | 2012-10-17 | 中山大学肿瘤防治中心 | MiRNA in-situ hybridization probe, its detection kit and its application |
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| CN102952848A (en) * | 2011-08-17 | 2013-03-06 | 上海交通大学医学院附属第三人民医院 | Detection method of small non-messenger RNA |
| CN102952848B (en) * | 2011-08-17 | 2015-09-23 | 上海交通大学医学院附属第三人民医院 | A kind of method detecting non-coding tiny RNA |
| CN102382893A (en) * | 2011-11-23 | 2012-03-21 | 博生吉医药科技(苏州)有限公司 | Specificity quantitive detection for cell apoptosis |
| CN102533984A (en) * | 2011-12-19 | 2012-07-04 | 苏州福英基因科技有限公司 | Horizontal in-situ hybridization detection kit and detection method as well as application for MICRORNA17-3P in earlier stage of cancer pathologic evolution |
| CN102732619A (en) * | 2012-06-06 | 2012-10-17 | 中山大学肿瘤防治中心 | MiRNA in-situ hybridization probe, its detection kit and its application |
| CN107406877A (en) * | 2015-02-04 | 2017-11-28 | 博洛尼亚大学 | For accelerating the additive of hybridization reaction |
| CN107406877B (en) * | 2015-02-04 | 2021-06-18 | 博洛尼亚大学 | Additive for accelerating hybridization reaction |
| CN106841135A (en) * | 2017-01-06 | 2017-06-13 | 东南大学 | A kind of method that various miRNA are detected by fluorescence method simultaneously |
| CN106841135B (en) * | 2017-01-06 | 2019-05-31 | 东南大学 | A method of a variety of miRNA are detected by fluorescence method simultaneously |
| CN111363789A (en) * | 2018-12-25 | 2020-07-03 | 中山大学孙逸仙纪念医院 | Kit and method for simultaneously detecting protein and RNA |
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