CN114645076B - miR-589-3p horizontal in-situ hybridization detection kit and detection method - Google Patents

miR-589-3p horizontal in-situ hybridization detection kit and detection method Download PDF

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CN114645076B
CN114645076B CN202210192180.2A CN202210192180A CN114645076B CN 114645076 B CN114645076 B CN 114645076B CN 202210192180 A CN202210192180 A CN 202210192180A CN 114645076 B CN114645076 B CN 114645076B
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situ hybridization
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probe
hybridization
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CN114645076A (en
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廖兴华
沈超
李佳蓬
项园
戴周彤
张慧敏
王君
黄优
宗启贝
李乐威
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Wuhan University of Science and Engineering WUSE
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Abstract

The invention provides a miR-589-3p horizontal in-situ hybridization detection kit and a detection method, wherein the kit comprises a CD30 monoclonal antibody, a miR-589-3p probe, a sealing liquid, a fixing liquid, a hybridization liquid and a counterstain. The invention adopts the CD30 immunofluorescence antibody and miR-589-3p fluorescence in situ hybridization probe to detect the cancer lesion condition, and solves the technical problems of false positive and poor penetrating power in fluorescence in situ hybridization detection.

Description

miR-589-3p horizontal in-situ hybridization detection kit and detection method
Technical Field
The invention relates to the technical field of medicines, in particular to a miR-589-3p horizontal in-situ hybridization detection kit and a detection method.
Background
Fluorescent in situ hybridization (Fluorescence in situ hybridization, FISH) is an emerging molecular cytogenetic technology, a nonradioactive in situ hybridization technology developed based on the original radioactive in situ hybridization technology at the end of the 80 s of the 20 th century. The technology is widely applied to a plurality of fields such as animal and plant genome structure research, chromosome fine structure variation analysis, virus infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research.
The basic principle of FISH is that known labeled single-stranded nucleic acid is used as a probe, and the known labeled single-stranded nucleic acid is combined with unknown single-stranded nucleic acid in a material to be detected in a specific way according to the base complementation principle to form the hybridization double-stranded nucleic acid which can be detected. Since DNA molecules are arranged linearly along the longitudinal axis of the chromosome on the chromosome, probes can be hybridized directly to the chromosome to localize a specific gene on the chromosome. Compared with the traditional radiolabeled in situ hybridization, the fluorescent in situ hybridization has the characteristics of rapidness, strong detection signal, high hybridization specificity, capability of multiple staining and the like, and therefore, the fluorescent in situ hybridization is widely focused in the field of molecular cytogenetics.
MicroRNA (miRNA) is an endogenous non-coding small molecular RNA with the length of about 18-25 nucleotides, and is regulated and controlled to express mRNA by two main mechanisms of inhibiting translation or accelerating degradation of target messenger RNAs (mRNAs) through combining with a 3 'untranslated region (3' UTR) of the mRNAs, so that important roles in biological development, occurrence and development of diseases and the like are played, and the roles in tumors are also becoming more and more important. The study on miR-589 is mainly aimed at miR-589-5P, and the study on miR-589-5P adopts high-throughput gene chip analysis technologies such as Northern hybridization, an expression chip, real-time fluorescence quantitative PCR, solexa sequencing and the like, such as: the expression quantity of miR-589-5P is in negative correlation in liver cancer tissues, and miR-589-5P can be used for diagnosing acute gout. There is no study of the application of miR-589-3P level in situ hybridization in the detection of precancerous lesions. Meanwhile, the existing widely used miRNA fluorescence in-situ hybridization has the technical problems of false positive caused by poor self fluorescence penetrability and reduced detection sensitivity.
Disclosure of Invention
In view of the above, the invention provides a miR-589-3p level in-situ hybridization detection kit and a detection method capable of reducing false positive and improving detection sensitivity.
The technical scheme of the invention is realized as follows: the invention provides a miR-589-3p horizontal in-situ hybridization detection kit, which is characterized in that: comprises CD30 monoclonal antibody, miR-589-3p probe, hybridization solution, fixing solution, blocking solution and counterstain.
Based on the technical scheme, preferably, the CD30 monoclonal antibody is a fluorescence labeled CD30 monoclonal antibody, the miR-589-3p probe is a miR-589-3p fluorescence in-situ hybridization probe, and the nucleotide sequence of the miR-589-3p fluorescence in-situ hybridization probe is shown as SEQ ID NO. 1.
On the basis of the technical scheme, preferably, the hybridization solution comprises dextran sulfate with the mass fraction of 15-30%, formamide with the concentration of 2-5mol/L, biological guanidine isothiocyanate with the concentration of 1-2mol/L, 6 XSSC and Triton-100 with the volume fraction of 1-3%, and the solvent is deionized water.
On the basis of the technical scheme, preferably, the fixing liquid comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
On the basis of the technical scheme, preferably, the blocking solution comprises 1-3% of BSA by mass, 3-5% of Tween20 by mass and 10 XSSC by mass, and the solvent is deionized water.
On the basis of the technical scheme, preferably, the counterstain is PI/anti-end or DAPI/anti-end dye liquor.
The invention also provides a using method of the miR-589-3p horizontal in-situ hybridization detection kit, which comprises the following steps:
s1, tabletting: transferring the harvested cell sample into a centrifuge tube, centrifuging, discarding the supernatant, performing hypotonic, pre-fixing and fixing operations to prepare cell suspension, sucking the cell suspension to a glass slide, and aging to obtain a cell slice;
s2, hybridization pretreatment: the cell slide is sealed, perforated and dehydrated to obtain a dry slide;
s3, denaturation treatment: the miR-589-3p fluorescent in-situ hybridization probe and the cell slide are subjected to denaturation treatment at 70-80 ℃;
s4, hybridization incubation: uniformly mixing the fluorescence-labeled CD30 monoclonal antibody, the denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, then dripping the mixture into a denatured cell slide hybridization area, then covering a cover glass, and carrying out hybridization incubation after edge sealing;
s5, washing: after incubation, eluting the slide for multiple times, soaking in deionized water, and naturally drying;
s6, sealing piece observation: the counterstain was added dropwise to the dried slide, and the slide was observed under a fluorescence microscope.
Based on the above technical solution, preferably, the denaturation treatment in step S3 is: immersing the cell slide in a denaturing solution of formamide/2 XSSC with the volume fraction of 70% at 70-80 ℃ for 2-3min; incubating miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 70-80 ℃ for 5min, and then placing at 0 ℃ for 5-10 min to denature the double-stranded DNA probe.
Compared with the prior art, the miR-589-3p horizontal in-situ hybridization detection kit and the detection method have the following beneficial effects: the invention adopts the CD30 immunofluorescence antibody and miR-589-3p fluorescence in situ hybridization probe to detect the cancer lesion condition, and solves the technical problems of false positive and poor penetrating power in fluorescence in situ hybridization detection. The kit can quickly complete hybridization within 2 hours, and the hybridization time is greatly shortened compared with 16-24 hours of the traditional probe. The kit can detect the expression quantity of miR-589-3p genes on the mRNA level, and can realize the screening of the mRNA level of various precancerous lesions.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a microscopic image of lung cancer positives detected by in situ hybridization according to an embodiment;
FIG. 2 is a schematic representation of a second embodiment of in situ hybridization detection of breast cancer positive microscopy;
FIG. 3 is a three-position hybridization detection gastric cancer positive microscopic examination chart of the embodiment;
FIG. 4 is a microscopic image of gastric cancer positivity detected by in situ hybridization of comparative example.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
The invention firstly provides a miR-589-3p horizontal in-situ hybridization detection kit, which comprises a CD30 monoclonal antibody, a miR-589-3p probe, a blocking solution, a fixing solution, a hybridization solution, an eluent and a counterstain.
The CD30 monoclonal antibody is fluorescence marked CD30 monoclonal antibody; the miR-589-3p probe is a miR-589-3p fluorescent in-situ hybridization probe, and the nucleotide sequence of the miR-589-3p fluorescent in-situ hybridization probe is shown as SEQ ID NO. 1.
The hybridization solution comprises dextran sulfate with the mass fraction of 15% -30%, formamide with the concentration of 2-5mol/L, biological guanidine isothiocyanate with the concentration of 1-2mol/L, 6 XSSC and Triton-100 with the volume fraction of 1-3%, and the solvent is deionized water.
The fixing solution comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
The perforator included Triton X-100 at a mass fraction of 0.5% and BSA at a mass fraction of 3%.
The blocking solution comprises 1-3% of BSA by mass, 3-5% of Tween20 by mass and 10 XSSC by mass, and the solvent is deionized water.
The counterstain is PI/anti-end or DAPI/anti-end dye liquor.
Example 1
The application method of the miR-589-3p horizontal in-situ hybridization detection kit uses a lung cancer cell sample as a detection specimen. The method comprises the following steps:
s1, tabletting: sucking the captured lung cancer cell sample into a sharp-bottomed centrifuge tube, centrifuging at 2500 rpm for 10min, and removing the supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, and the mixture is blown and evenly mixed and then placed in a 37 ℃ incubator for 30min; adding 2mL of fixing solution, blowing, mixing uniformly, and centrifuging for 10min at 2500 rpm; sucking the supernatant, adding 5mL of fixing solution, blowing and mixing uniformly, fixing for 10min, centrifuging for 10min at 2500 rpm, and repeating the steps until the cell sediment is washed completely; then sucking the supernatant, adding a proper amount of fixing solution, and preparing a cell suspension with proper concentration; then sucking 5 mu L of cell suspension liquid to a glass slide, and aging for 1 hour at 60 ℃ to obtain lung cancer cell tabletting; the fixing solution is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 2:1.
S2, pre-hybridization slide treatment: 100ul of blocking solution is added to a lung cancer cell slide, and after blocking for 10min at room temperature, 2 XSSC (pH 7.0) is rinsed for 5min; then 100ul of perforating agent is dripped, the room temperature is closed for 10min, and 2 XSSC (pH 7.0) is rinsed for 5min; sequentially placing the slide in 70Vt% ethanol, 85Vt% ethanol and 100Vt% ethanol for 2min, dehydrating, and naturally drying; the blocking solution comprises 1% of BSA by mass, 3% of Tween20 by mass and 10 XSSC by mass, and the solvent is deionized water. The perforator comprises Triton X-100 at a mass fraction of 0.2% and BSA at a mass fraction of 1%.
S3, denaturation treatment: immersing lung cancer cell slide in 70 ℃ formamide/2 XSSC denaturing solution with volume fraction of 70% for 2min; incubating miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 70 ℃ for 5min, and then placing at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, taking 10 mu L of the mixture to be dripped into a cell dripping sheet hybridization area, immediately covering a cover glass, uniformly spreading the probe under the cover glass without bubbles, sealing the edges by using resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises dextran sulfate 15% by mass, formamide with concentration of 2mol/L, biological guanidine isothiocyanate with concentration of 1mol/L, 6 XSSC and Triton-100 with volume fraction of 1%, and the solvent is deionized water
S5, washing: taking out the slide specimen from the incubator, and gently removing the cover glass by using a blade; then placing in preheated 40 ℃ formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5min; then washing 3 times in 1 XSSC preheated to 40℃for 5min each; slide specimens were washed in deionized water at room temperature. Taking out the slide, and naturally drying.
S6, sealing piece observation: 200. Mu.L of counterstain solution PI/antifade was added dropwise to the slide specimen, covered with a coverslip, and the slide was observed under a fluorescence microscope, and the result was shown in FIG. 1.
Example two
The application method of the miR-589-3p horizontal in-situ hybridization detection kit adopts a breast cancer cell sample as a detection specimen. The method comprises the following steps:
s1, tabletting: sucking the captured breast cell sample into a sharp-bottomed centrifuge tube, centrifuging at 2500 rpm for 10min, and removing the supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, and the mixture is blown and evenly mixed and then placed in a 37 ℃ incubator for 30min; adding 2mL of fixing solution, blowing, mixing uniformly, and centrifuging for 10min at 2500 rpm; sucking the supernatant, adding 5mL of fixing solution, blowing and mixing uniformly, fixing for 10min, centrifuging for 10min at 2500 rpm, and repeating the steps until the cell sediment is washed completely; then sucking the supernatant, adding a proper amount of fixing solution, and preparing a cell suspension with proper concentration; then sucking 5 mu L of cell suspension liquid to a glass slide, and aging for 1 hour at 60 ℃ to obtain a breast cancer cell slice; the fixing solution is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 5:1.
S2, pre-hybridization slide treatment: 100ul of blocking solution is added to the breast cancer cell slide, and after blocking for 10min at room temperature, 2 XSSC (pH 7.0) is rinsed for 5min; then 100ul of perforating agent is dripped, the room temperature is closed for 10min, and 2 XSSC (pH 7.0) is rinsed for 5min; sequentially placing the slide in 70Vt% ethanol, 85Vt% ethanol and 100Vt% ethanol for 2min, dehydrating, and naturally drying; the blocking solution comprises 3% of BSA by mass, 5% of Tween20 by mass and 10 XSSC by mass, and the solvent is deionized water. The perforator included Triton X-100 at a mass fraction of 0.5% and BSA at a mass fraction of 3%.
S3, denaturation treatment: immersing a breast cancer cell slide in a formamide/2 XSSC denaturing solution with a volume fraction of 70% at 80 ℃ for 2min; incubating miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 80 ℃ for 5min, and then placing at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, taking 10 mu L of the mixture to be dripped into a cell dripping sheet hybridization area, immediately covering a cover glass, uniformly spreading the probe under the cover glass without bubbles, sealing the edges by using resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises dextran sulfate 30% by mass, formamide with concentration of 5mol/L, biological guanidine isothiocyanate with concentration of 2mol/L, 6 XSSC and Triton-100 with volume fraction of 3%, and the solvent is deionized water
S5, washing: taking out the slide specimen from the incubator, and gently removing the cover glass by using a blade; then placing in preheated 40 ℃ formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5min; then washing 3 times in 1 XSSC preheated to 40℃for 5min each; slide specimens were washed in deionized water at room temperature. Taking out the slide, and naturally drying.
S6, sealing piece observation: 200. Mu.L of counterstain solution DAPI/anilade was added dropwise to the slide specimen, covered with a coverslip, and the slide was observed under a fluorescence microscope, and the result is shown in FIG. 2.
Example III
The application method of the miR-589-3p horizontal in-situ hybridization detection kit uses a gastric cancer cell sample as a detection specimen. The method comprises the following steps:
s1, tabletting: sucking the captured gastric cancer cell sample into a sharp-bottomed centrifuge tube, centrifuging at 2500 rpm for 10min, and removing the supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, and the mixture is blown and evenly mixed and then placed in a 37 ℃ incubator for 30min; adding 2mL of fixing solution, blowing, mixing uniformly, and centrifuging for 10min at 2500 rpm; sucking the supernatant, adding 5mL of fixing solution, blowing and mixing uniformly, fixing for 10min, centrifuging for 10min at 2500 rpm, and repeating the steps until the cell sediment is washed completely; then sucking the supernatant, adding a proper amount of fixing solution, and preparing a cell suspension with proper concentration; then sucking 5 mu L of cell suspension liquid to a glass slide, and aging for 1 hour at 60 ℃ to obtain a gastric cancer cell slice; the fixing solution is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 3:1.
S2, pre-hybridization slide treatment: 100ul of blocking solution is added to the gastric cancer cell slide, and after blocking for 10min at room temperature, 2 XSSC (pH 7.0) is rinsed for 5min; then 100ul of perforating agent is dripped, the room temperature is closed for 10min, and 2 XSSC (pH 7.0) is rinsed for 5min; sequentially placing the slide in 70Vt% ethanol, 85Vt% ethanol and 100Vt% ethanol for 2min, dehydrating, and naturally drying; the blocking solution comprises 2% of BSA by mass, 4% of Tween20 by mass and 10 XSSC by mass, and the solvent is deionized water. The perforator included Triton X-100 at a mass fraction of 0.3% and BSA at a mass fraction of 2%.
S3, denaturation treatment: immersing a gastric cancer cell slide in a formamide/2 XSSC denaturing solution with a volume fraction of 70% at 80 ℃ for 2min; incubating miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 80 ℃ for 5min, and then placing at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, taking 10 mu L of the mixture to be dripped into a cell dripping sheet hybridization area, immediately covering a cover glass, uniformly spreading the probe under the cover glass without bubbles, sealing the edges by using resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises dextran sulfate with the mass fraction of 20%, formamide with the concentration of 4mol/L, biological guanidine isothiocyanate with the concentration of 1.5mol/L, 6 XSSC and Triton-100 with the volume fraction of 2%, and the solvent is deionized water;
s5, washing: taking out the slide specimen from the incubator, and gently removing the cover glass by using a blade; then placing in preheated 40 ℃ formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5min; then washing 3 times in 1 XSSC preheated to 40℃for 5min each; slide specimens were washed in deionized water at room temperature. Taking out the slide, and naturally drying.
S6, sealing piece observation: 200 mu L of counterstain solution DAPI/antifade is dripped on a slide specimen, a cover glass is covered, and the slide is observed under a fluorescence microscope; the results are shown in FIG. 3.
The comparative example uses the existing fluorescence in situ hybridization method as a comparative example I, no antibody is generated, only a probe is hybridized with cells, and 80 cases are detected in each example, wherein 50 cases are positive, the test sample of the comparative example I is the same as that of the example I, and the test result is as follows:
TABLE 1 cancer detection results
As shown in Table 1, the invention adopts the CD30 immunofluorescence antibody and miR-589-3p fluorescence in situ hybridization probe to detect cancer lesions, overcomes the technical problems of false positive and poor penetrability in fluorescence in situ hybridization detection, and can rapidly complete hybridization within 2 hours, thereby greatly shortening detection time.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> university of Wuhan science and technology
<120> miR-589-3p horizontal in situ hybridization detection kit and detection method
<141> 2022-02-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 106
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccgttcagaa caaatgccgg ctgttaatgc taatcgtgat aggggttttt gcctccaact 60
gactcctaca tattagcatt aacagagtgc agggtccgag gtattc 106

Claims (7)

  1. miR-589-3p level in situ hybridization detection kit is characterized in that: comprises CD30 monoclonal antibody, miR-589-3p probe, hybridization solution, fixing solution, blocking solution and counterstain; the CD30 monoclonal antibody is a fluorescence-marked CD30 monoclonal antibody, the miR-589-3p probe is a miR-589-3p fluorescence in-situ hybridization probe, and the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
  2. 2. The miR-589-3p level in situ hybridization assay kit of claim 1, wherein: the hybridization solution comprises dextran sulfate with the mass fraction of 15-30%, formamide with the concentration of 2-5mol/L, biological guanidine isothiocyanate with the concentration of 1-2mol/L, 6 XSSC and Triton-100 with the volume fraction of 1-3%, and the solvent is deionized water.
  3. 3. The miR-589-3p level in situ hybridization assay kit of claim 1, wherein: the fixing solution comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
  4. 4. The miR-589-3p level in situ hybridization assay kit of claim 1, wherein: the blocking solution comprises 1-3% of BSA by mass fraction, 3-5% of Tween20 by mass fraction and 10 XSSC by mass fraction, and the solvent is deionized water.
  5. 5. The miR-589-3p level in situ hybridization assay kit of claim 1, wherein: the counterstain is PI/anti-end or DAPI/anti-end dye liquor.
  6. 6. The application method of the miR-589-3p level in-situ hybridization detection kit for non-disease diagnosis is characterized by comprising the following steps of: the kit is the kit as claimed in any one of claims 1 to 5, and the use method comprises the following steps:
    s1, tabletting: transferring the harvested cell sample into a centrifuge tube, centrifuging, discarding the supernatant, performing hypotonic, pre-fixing and fixing operations to prepare cell suspension, sucking the cell suspension to a glass slide, and aging to obtain a cell slice;
    s2, hybridization pretreatment: the cell slide is sealed, perforated and dehydrated to obtain a dry slide;
    s3, denaturation treatment: the miR-589-3p fluorescent in-situ hybridization probe and the cell slide are subjected to denaturation treatment at 70-80 ℃;
    s4, hybridization incubation: uniformly mixing the fluorescence-labeled CD30 monoclonal antibody, the denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, then dripping the mixture into a denatured cell slide hybridization area, then covering a cover glass, and carrying out hybridization incubation after edge sealing;
    s5, washing: after the incubation is finished, eluting the cell slide for a plurality of times, soaking the cell slide in deionized water, and naturally drying the cell slide;
    s6, sealing piece observation: the counterstain was added dropwise to the dried cell slide and the slide was observed under a fluorescence microscope.
  7. 7. The method for using the miR-589-3p level in situ hybridization detection kit for non-disease diagnosis according to claim 6, wherein: the denaturation treatment in step S3 is as follows: immersing the cell slide in a 70-80 ℃ formamide/2 XSSC denaturing solution with a volume fraction of 70% for 2-3min; incubating miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 70-80 ℃ for 5min, and then placing the probe at 0 ℃ for 5-10 min to denature the double-stranded DNA probe.
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