CN109988846A - A method of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization - Google Patents
A method of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization Download PDFInfo
- Publication number
- CN109988846A CN109988846A CN201910178865.XA CN201910178865A CN109988846A CN 109988846 A CN109988846 A CN 109988846A CN 201910178865 A CN201910178865 A CN 201910178865A CN 109988846 A CN109988846 A CN 109988846A
- Authority
- CN
- China
- Prior art keywords
- situ hybridization
- paraffin section
- probe
- mrna
- red claw
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
-
- 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
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization, belong to hybridization in situ technique field, it including gonadal tissue embedding, paraffin section, in situ hybridization and photographs to record, wherein in situ hybridization uses probe sequence for as shown in SEQ ID NO.3.Specific specificity is located to the tissue of dsx gene in probe of the present invention, realizes the effect of the display to dsx gene mRNA levels；The present invention establishes the method for paraffin section mRNA in situ hybridization in red claw crayfish to study expression pattern of the related gene in sexual gland, can clearly describe expression and localization of the red claw crayfish gender related gene in sexual gland；The method of the present invention avoids tissue contracts and cytomorphosis while paraffin section de-waxing effect is good, so that tissue remains at original position, tissue morphology keeps complete, hydrolysis result can also be improved, so that cell color is good, caryoplasm is clearly demarcated, the final accuracy for improving in situ hybridization.
Description
Technical field
The invention belongs to hybridization in situ technique fields, and in particular to one kind is suitable for red claw crayfish gonadal tissue mRNA paraffin and cuts
The method of piece in situ hybridization.
Background technique
Red claw crayfish (Cherax quadricariratus), is commonly called as Australia freshwater lobster, originates in the northern heat of Australia
Region and New Guinea south, are under the jurisdiction of bare hull shrimp category, have and grow the spies such as fast, degeneration-resistant strong, fine and tender taste, dressing percentage height
Point is one of rare economic shrimps of world's fresh water.As other fresh water decapods animals, red claw crayfish male is than female
Grow faster, figure it is also bigger.Extensive cultivation has been obtained especially nearly ten years due to its biology in red chela chela traitor
Property comparison is obvious, and the speed of the aquaculture development of red chela chela traitor is quickly.However, at present for the shrimp property phenotypic differentiation and two
The mechanism of state property development is unclear.In situ hybridization tissue (or cell) chemistry (In situ Hybridization
Histochemistry, ISHH) abbreviation in situ hybridization (In Situ Hybridization), belong to the model of solid phase molecules hybridization
Farmland, it is probe that it, which is with the DNA of label or RNA, and the method for detecting specific nucleic acid sequence in histocyte in situ is widely applied
In distribution of the lab analysis mRNA in biological tissue and enrichment degree.Therefore it establishes and is suitable for red claw crayfish gonadal tissue
Paraffin section mRNA hybridization in situ technique carries out tissue positioning and functional study to Sex Determination related gene, helps speed up
The molecular mechanism of action of red claw crayfish Sex determination and differentiation is disclosed and illustrates, to push crayfish sexual control breeding technique
Development.
Summary of the invention
Specific specificity is shown the purpose of the present invention is to provide a kind of pair of dsx gene, is realized to dsx gene
The method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe of the display effect of mRNA level in-site.
The technical solution that the present invention is taken to achieve the above object are as follows:
The present invention provides a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, presses
According to following steps:
S1 step: the red claw crayfish gonadal tissue cDNA obtained using reverse transcription is reacted by PCR using primer and is expanded as template
Increasing obtains the part ORF sequence of dsx gene, the sequence probe sequence special as dsx gene mRNA；
S2 step: the probe plasmid of building dsx gene mRNA in situ hybridization, after probe plasmid is converted bacterium, bacterium training
It is sequenced after supporting；
S3 step: preparation dsx gene mRNA in situ hybridization probe.The double property genes of the Sex Determination of red claw crayfish are all dsx
Homologous gene, double property gene dsx are in Sex Determination signal path most downstream, and upstream relative Sex Determination important gene is come
It says, quite conservative, variation is only that it splices form；And regulate and control the splicing enhancing spliced or inhibiting factor and therewith phase
In conjunction with transacting element, almost do not change in same suborder, it is regular between different suborders to follow.
Preferably, dsx gene is expressed in spermary, and is not expressed in ovary.
Preferably, primer
Forward primer are as follows: 5 '-GAATTCTAATACGACTCACTATAGGGA-3 '；
Reverse primer are as follows: 5 '-CCTATAGTGAGTCGTATTAAAGCTT-3 '.
The display that shows specific specificity, realize to dsx gene mRNA levels of the probe of the present invention to dsx gene
Effect.
One kind being suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, is made using the above method.
Preferably, probe sequence is as shown in SEQ ID NO.3.
It is another object of the present invention to provide a kind of paraffin section de-waxing effect is good, hydrolysis result is good, hybridization signal
By force, cell color is good, can clearly describe to stick up expression and localization of the mouth Culter gender related gene in sexual gland, accuracy is high to fit
In the method for red claw crayfish gonadal tissue mRNA frozen section in situ hybridization.
The technical solution that the present invention is taken to achieve the above object are as follows:
The present invention provides a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization, including sexual gland
It organization embedding, paraffin section, in situ hybridization and photographs to record, in situ hybridization uses above-mentioned probe.
Preferably, in situ hybridization includes the following steps:
1) paraffin section de-waxing: slice is successively washed with dimethylbenzene, dehydrated alcohol, 85% alcohol, 75% alcohol, DEPC
It dewaxes；
2) it digests: slice after washing being boiled in reparation liquid, gene stroke circle after natural cooling, not according to different tissues
Protease K digesting is added dropwise in same index properties, rinses；
3) prehybridization: prehybridization solution will be added dropwise on slice after digestion, is incubated for；
4) hybridize: incline prehybridization solution, and hybridization solution containing probe is added dropwise, washs after hybridized overnight；
3) it closes: closing serum BSA is added dropwise, incline deblocking liquid, and anti-DIG-HRP is then added dropwise, and is incubated for；
4) DAB develops the color: after slice slightly dries, the DAB developing solution of Fresh being added dropwise in circle, controls colour developing under microscope
Time, the positive are brown color, and pure water rinsing is sliced color development stopping；
5) it redyes nucleus: being redyed with haematoxylin dye liquor, broken up after washing through 1% hydrochloride alcohol, washing, ammonium hydroxide returns indigo plant,
It rinses；
6) mounting: neutral gum mounting.
Preferably, 0.03-0.06% trichloroacetaldehyde and 2.8-3.0% benzoin alcohol are contained in dimethylbenzene.Trichloroacetaldehyde and benzene
On the one hand the presence of fluorenol promotes toluene quickly to dissolve the paraffin for being used to support tissue and cell, good in paraffin section de-waxing effect
While avoid tissue contracts and cytomorphosis so that tissue remain at original position, tissue morphology has kept complete；On the other hand
The permeability that antibody on cell film can be increased, during DAB colour developing and haematoxylin dye liquor are redyed, so that cell color
Good, caryoplasm is clearly demarcated, clear in structure, and tissue, cell are more transparent clear, convenient for observation, improves the accuracy of in situ hybridization；This
Outside, moreover it is possible to which the toughness for increasing DNA chain avoids DNA chain and protein from forming compound and hinders the digestion of protease, improves enzymatic hydrolysis
Effect, the final accuracy for improving in situ hybridization.
Preferably, the concentration of probe hybridization solution is 50ng/ul.
Preferably, gonadal tissue includes the following steps:
Tissue is taken out to be immediately placed in fixer after cleaning and fixes 2-12h, after fixed completion after gradient alcohol dehydration
Waxdip, embedding.
Compared with prior art, the invention has the benefit that
The display that shows specific specificity, realize to dsx gene mRNA levels of the probe of the present invention to dsx gene
Effect；The present invention establishes the method for paraffin section mRNA in situ hybridization in red claw crayfish to study related gene in sexual gland
Expression pattern, expression and localization of the red claw crayfish gender related gene in sexual gland can clearly be described, for sex phenotype
The identification of gene has great importance；The method of the present invention avoids tissue contracts and thin while paraffin section de-waxing effect is good
Born of the same parents' deformation, so that tissue remains at original position, tissue morphology has kept complete, moreover it is possible to hydrolysis result is improved, so that cell color
Good, caryoplasm is clearly demarcated, the final accuracy for improving in situ hybridization.
One kind, which is provided, present invention employs above-mentioned technical proposal is suitable for red claw crayfish gonadal tissue mRNA paraffin section original position
The method of hybridization compensates for the deficiencies in the prior art, reasonable design, easy operation.
Detailed description of the invention
Fig. 1 is paraffin section situ Analysis of the red claw crayfish dsx gene in gonadal tissue in the embodiment of the present invention 1
Schematic diagram.
Specific embodiment
In the following, being suitable for red claw crayfish gonadal tissue mRNA stone to one kind of an embodiment of the present invention in conjunction with specific embodiments
The method of wax slice in situ hybridization is described further.
Embodiment 1:
A method of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, according to following step
It is rapid:
S1 step: positive and negative according to the primer sequence for sticking up mouth Culter dsx coding sequence design construction dsx probe carrier
It is respectively provided with EcoRI and Xhol restriction enzyme site to primer, as follows:
Forward primer are as follows: 5 '-GAATTCTAATACGACTCACTATAGGGA-3 ',
Reverse primer are as follows: 5 '-CCTATAGTGAGTCGTATTAAAGCTT-3 '；
S2 step: the red claw crayfish gonadal tissue cDNA obtained using reverse transcription is reacted by PCR using primer and is expanded as template
Increasing obtains the part ORF sequence of dsx gene, the sequence probe sequence special as dsx albumen, and PCR method is 94 DEG C of 4min；
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 32cycle；72 DEG C of extension 7min, utilize Axygen Gel Extraction kit mesh
Segment；
S3 step: target fragment and pBluescriptII SK carrier are subjected to double digestion processing with EcoRI and Xhol, instead
Answer system as shown in table 1, add and mix sufficiently after reagent, digest 4h in 37 DEG C of water-baths, after purified；
1 double enzyme digestion reaction system of table
| Component | Volume |
| Target fragment or carrier | 3μg |
| 10×H Buffer | 7μl |
| EcoRI | 3.5μl |
| Xhol | 3.5μl |
| ddH2O | It is supplemented to 70 μ l |
S4 step: by after digestion carrier and target fragment connected in 16 DEG C of water-baths, then conversion, sequencing, pass through sequence
Column analyze and identify correct recombinant plasmid；
S5 step: selection EcoRI or Xhol carries out single endonuclease digestion, 37 DEG C of water-bath digestion 4h, the weight linearized to plasmid
Group plasmid, and purified with the Cleanup kit of Axygen.Then as template, transcribed strand digoxigenin labeled it is anti-
Adopted rna probe, specific system is as shown in table 2, is uniformly mixed after adding reagent, and then 2 μ l are added in 37 DEG C of water-bath 2.5h
The DNaseI digested plasmid template of RNA enzyme free, 37 DEG C of incubation 20min, is added the 0.2M EDTA of 2 μ l later, stand on ice to
Digestion reaction terminates；
Table 2 transcribes rna probe reactant
S6 step: after digestion, rna probe is carried out using Mini Quick Spin RNA Columns (Roche)
Purifying, and electrophoresis and Concentration Testing are carried out to purified product, isometric deionized formamide is added in last remaining sample, mixed point light
Dress is placed on -80 DEG C of preservations, and the mRNA in situ hybridization probe of dsx gene is made.The double property genes of the Sex Determination of red claw crayfish are all
It is the homologous gene of dsx, double property gene dsx are in Sex Determination signal path most downstream, the important base of upstream relative Sex Determination
Quite conservative because for, variation is only that it splices form；And regulation splicing splicing enhancing or inhibiting factor and with
The transacting element combined, almost do not change in same suborder, it is regular between different suborders to follow.
Above-mentioned dsx gene is expressed in spermary, and is not expressed in ovary.
One kind being suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, is made using the above method.
Above-mentioned probe sequence be as shown in SEQ ID NO.3, specifically:
GAATTCTAATACGACTCACTATAGGGATGGCTATGGCGCCTAAGAAGAAATTCGATTTTTTCAATGGT
GGTCGGGGAATTGTCCTGACCGACACAACTGATGTGGAAGAATCGTCTGTAGAGCAGCAGCCACCAGAGCCCAAGG
TAGTGCAGGAGTCTTATGGTTATGGAGGCGACTATGGGCGCTACTCTGGTTCTTCCGAGGGCTACGTAACCCCCAG
CTCTCCTGGGGGCTATATGCCTCCAGGGGGCTATGTCCCCCCCAGATCTCCAGGGGGCTATGTCGTCCCCAGATCT
CCAGGGGGCTGTGTGTCCTCTAGATCTCCAGGGGAGTATGTGCCCCCTGACAGCCCGAATGTTCATCAGTACCACG
GTGACCCTATAGTGAGTCGTATTAAAGCTT。
Embodiment 2:
A method of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization, including gonadal tissue embedding,
It paraffin section, in situ hybridization and photographs to record, in situ hybridization uses above-mentioned probe.
Above-mentioned in situ hybridization includes the following steps:
1) tissue is fixed: tissue taking-up is immediately placed in fixer (preparation of DEPC water) after cleaning and fixes 2-12h；
2) it is dehydrated: waxdip, embedding after gradient alcohol dehydration after the fixed completion of tissue；
3) be sliced: the sliced machine-cut piece of paraffin, booth piece machine fish out piece, and 62 DEG C of ovens bake piece 2h；
4) paraffin section de-waxing is to water: slice being successively put into I 15min- dimethylbenzene of dimethylbenzene, II 15min- dehydrated alcohol I
II 5min-85% alcohol 5min-75% alcohol 5min-DEPC of 5min- dehydrated alcohol washing；
5) digest: according to tissue set time length, slice boils 10-15 minutes in reparation liquid, base after natural cooling
Because Proteinase K (20ug/ml) 37 DEG C of digestion 30min, pure water rinsing is added dropwise according to different tissues difference index properties in stroke circle
PBS washes 3 times × 5min afterwards；
6) prehybridization solution, 37 DEG C of incubation 1h prehybridization: are added dropwise；
7) hybridize: incline prehybridization solution, and hybridization solution containing probe, concentration 50ng/ul is added dropwise, and 37 DEG C of degree of insulating box hybridized
Night；
8) post-hybridization washing: washing away hybridization solution, 2 × SSC, washes 10min, 1 × SSC, 37 DEG C for 37 DEG C and washes 2 × 5min, 0.5 ×
SSC room temperature washes 10min, if not specific hybrid is more, can increase formamide washing；
9) confining liquid is added dropwise: closing serum BSA, room temperature 30min is added dropwise；
10) the anti-digoxigenin labeled peroxidase (anti-DIG-HRP) of mouse is added dropwise: incline deblocking liquid, and anti-is added dropwise
DIG-HRP.37 DEG C of incubations 40min, rear PBS wash 4 times × 5min；
11) DAB develops the color: after slice slightly dries, the DAB developing solution of Fresh being added dropwise in circle, controls under microscope aobvious
Color time, the positive are brown color, and pure water rinsing is sliced color development stopping；
12) redye nucleus: haematoxylin redyes 3min or so, originally washes, and 1% hydrochloride alcohol breaks up several seconds, tap water
It washes, ammonium hydroxide returns indigo plant, and flowing water rinses；
13) mounting: neutral gum mounting；
14) microscope inspection, Image Acquisition analysis, as a result as shown in Figure 1, left figure is spermary in Fig. 1, right figure is ovary, letter
Number in spermary, it follows that dsx gene signal in spermary is stronger, and fails to detect signal in ovary, show dsx gene
It expresses in spermary, and is not expressed in ovary.
Embodiment 3:
In order to improve paraffin section de-waxing effect, further prioritization scheme are as follows:
Paraffin section de-waxing contains 0.03-0.06% trichloroacetaldehyde and 2.8-3.0% benzene fluorenes into water step in dimethylbenzene
Alcohol.On the one hand the presence of trichloroacetaldehyde and benzoin alcohol promotes toluene quickly to dissolve the paraffin for being used to support tissue and cell, avoid
Tissue contracts and cytomorphosis, so that tissue remains at original position, tissue morphology has kept complete；On the other hand can increase anti-
Body is to permeability of cell membranes, and during DAB colour developing and haematoxylin dye liquor are redyed, so that cell color is good, caryoplasm is clearly demarcated,
Clear in structure, tissue, cell are more transparent clear, convenient for observation, improve the accuracy of in situ hybridization；Moreover it is possible to increase DNA
The toughness of chain avoids DNA chain and protein from forming compound and hinders the digestion of protease, improves hydrolysis result, final to improve
The accuracy of in situ hybridization.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Sequence table
<110>Zhejiang Institute of Fresh Water Aquatic Products
<120>a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaattctaat acgactcact ataggga 27
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctatagtga gtcgtattaa agctt 25
<210> 3
<211> 402
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaattctaat acgactcact atagggatgg ctatggcgcc taagaagaaa ttcgattttt 60
tcaatggtgg tcggggaatt gtcctgaccg acacaactga tgtggaagaa tcgtctgtag 120
agcagcagcc accagagccc aaggtagtgc aggagtctta tggttatgga ggcgactatg 180
ggcgctactc tggttcttcc gagggctacg taacccccag ctctcctggg ggctatatgc 240
ctccaggggg ctatgtcccc cccagatctc cagggggcta tgtcgtcccc agatctccag 300
ggggctgtgt gtcctctaga tctccagggg agtatgtgcc ccctgacagc ccgaatgttc 360
atcagtacca cggtgaccct atagtgagtc gtattaaagc tt 402
Claims (10)
1. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, which is characterized in that press
According to following steps:
S1 step: the red claw crayfish gonadal tissue cDNA obtained using reverse transcription is expanded as template, using primer by PCR reaction
To the part ORF sequence of dsx gene, the sequence probe sequence special as dsx gene mRNA；
S2 step: the probe plasmid of building dsx gene mRNA in situ hybridization, after probe plasmid is converted bacterium, after Bacteria Culture
It is sequenced；
S3 step: preparation dsx gene mRNA in situ hybridization probe.
2. according to claim 1 a kind of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe
Method, it is characterised in that: the dsx gene is expressed in spermary, and is not expressed in ovary.
3. according to claim 1 a kind of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe
Method, it is characterised in that: the primer
Forward primer are as follows: 5 '-GAATTCTAATACGACTCACTATAGGGA-3 '；
Reverse primer are as follows: 5 '-CCTATAGTGAGTCGTATTAAAGCTT-3 '.
4. one kind is suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe, it is characterised in that: use right
It is required that method described in 1 or 2 or 3 is made.
5. one kind according to claim 4 is suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization probe,
It is characterized by: the probe sequence is as shown in SEQ ID NO.3.
6. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization, including gonadal tissue embedding, stone
Wax slice and photographs to record in situ hybridization, it is characterised in that: the in situ hybridization uses probe as claimed in claim 4.
7. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization according to claim 6,
It is characterized by: the in situ hybridization includes the following steps:
1) paraffin section de-waxing: slice is successively carried out with dimethylbenzene, dehydrated alcohol, 85% alcohol, 75% alcohol, DEPC washing
Dewaxing；
2) it digests: slice after washing being boiled in reparation liquid, gene stroke circle after natural cooling refers to according to different tissues difference
Characteristic is marked, protease K digesting is added dropwise, is rinsed；
3) prehybridization: prehybridization solution will be added dropwise on slice after digestion, is incubated for；
4) hybridize: incline prehybridization solution, and hybridization solution containing probe is added dropwise, washs after hybridized overnight；
3) it closes: closing serum BSA is added dropwise, incline deblocking liquid, and anti-DIG-HRP is then added dropwise, and is incubated for；
4) DAB develops the color: after slice slightly dries, the DAB developing solution of Fresh being added dropwise in circle, when controlling colour developing under microscope
Between, the positive is brown color, and pure water rinsing is sliced color development stopping；
5) it redyes nucleus: being redyed with haematoxylin dye liquor, broken up after washing through 1% hydrochloride alcohol, washing, ammonium hydroxide returns indigo plant, rinses；
6) mounting: neutral gum mounting.
8. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization according to claim 7,
It is characterized by: containing 0.03-0.06% trichloroacetaldehyde and 2.8-3.0% benzoin alcohol in the dimethylbenzene.
9. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization according to claim 7,
It is characterized by: the concentration of the probe hybridization solution is 50ng/ul.
10. a kind of method suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization according to claim 6,
It is characterized by: the gonadal tissue includes the following steps:
Tissue is taken out and is immediately placed in fixer after cleaning and fixes 2-12h, it is fixed complete after after gradient alcohol dehydration waxdip,
Embedding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910178865.XA CN109988846B (en) | 2019-03-11 | 2019-03-11 | Method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910178865.XA CN109988846B (en) | 2019-03-11 | 2019-03-11 | Method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109988846A true CN109988846A (en) | 2019-07-09 |
| CN109988846B CN109988846B (en) | 2023-05-26 |
Family
ID=67129607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910178865.XA Active CN109988846B (en) | 2019-03-11 | 2019-03-11 | Method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109988846B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734964A (en) * | 2019-09-17 | 2020-01-31 | 武汉赛维尔生物科技有限公司 | hybridization buffer solution system suitable for bacterial FISH detection and in-situ hybridization method thereof |
| CN114645083A (en) * | 2022-02-15 | 2022-06-21 | 浙江省农业科学院 | PCR amplification primer, kit and identification method for rapidly identifying genetic sex of red swamp crayfish |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1759190A (en) * | 2002-11-14 | 2006-04-12 | 比奥根艾迪克Ma公司 | Absolute quantitation of nucleic acids by RT-PCR |
| CN103074331A (en) * | 2012-09-10 | 2013-05-01 | 中国人民解放军第四军医大学 | KCC2 gene cRNA in situ hybridization probe and design method thereof |
| CN103320505A (en) * | 2013-03-09 | 2013-09-25 | 北京安必奇生物科技有限公司 | Design and detection method for in-situ hybridization probe of HSD11B1 gene |
| CN104232645A (en) * | 2014-09-17 | 2014-12-24 | 中国农业大学 | Peroxidase gene dsRNA (double-stranded ribonucleic acid) and application of peroxidase gene dsRNA in control of sitobion avenae |
| CN106916886A (en) * | 2017-01-18 | 2017-07-04 | 中国人民解放军第四军医大学 | A kind of gene primer of dynamin-related proteins 1 and its preparation method of cRNA in situ hybridization probes for FISH |
-
2019
- 2019-03-11 CN CN201910178865.XA patent/CN109988846B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1759190A (en) * | 2002-11-14 | 2006-04-12 | 比奥根艾迪克Ma公司 | Absolute quantitation of nucleic acids by RT-PCR |
| US20060149484A1 (en) * | 2002-11-14 | 2006-07-06 | Allaire Normand E | Absolute quantitation of nucleic acids by rt-pcr |
| CN103074331A (en) * | 2012-09-10 | 2013-05-01 | 中国人民解放军第四军医大学 | KCC2 gene cRNA in situ hybridization probe and design method thereof |
| CN103320505A (en) * | 2013-03-09 | 2013-09-25 | 北京安必奇生物科技有限公司 | Design and detection method for in-situ hybridization probe of HSD11B1 gene |
| CN104232645A (en) * | 2014-09-17 | 2014-12-24 | 中国农业大学 | Peroxidase gene dsRNA (double-stranded ribonucleic acid) and application of peroxidase gene dsRNA in control of sitobion avenae |
| CN106916886A (en) * | 2017-01-18 | 2017-07-04 | 中国人民解放军第四军医大学 | A kind of gene primer of dynamin-related proteins 1 and its preparation method of cRNA in situ hybridization probes for FISH |
Non-Patent Citations (3)
| Title |
|---|
| LI等: ""Identification and characterization of a doublesex gene which regulates the expression of insulin-like androgenic gland hormone in Fenneropenaeus chinensis"", 《GENE》 * |
| 孟繁雯等: "海洋放线菌WBF16的分类鉴定", 《药物生物技术》 * |
| 李法君等: "RNAi在甲壳动物中的研究进展", 《水生生物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734964A (en) * | 2019-09-17 | 2020-01-31 | 武汉赛维尔生物科技有限公司 | hybridization buffer solution system suitable for bacterial FISH detection and in-situ hybridization method thereof |
| CN114645083A (en) * | 2022-02-15 | 2022-06-21 | 浙江省农业科学院 | PCR amplification primer, kit and identification method for rapidly identifying genetic sex of red swamp crayfish |
| CN114645083B (en) * | 2022-02-15 | 2023-06-13 | 浙江省农业科学院 | PCR amplification primer, kit and identification method for rapidly identifying genetic sex of red swamp crayfish |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109988846B (en) | 2023-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20150105298A1 (en) | Multi-oligomer in situ hybridization probes | |
| CN109988846A (en) | A method of suitable for red claw crayfish gonadal tissue mRNA paraffin section in situ hybridization | |
| CN101440399A (en) | Molecular marking method for indicating and identifying litter size in pigs by MMP23 gene | |
| CN110964798B (en) | Method for identifying genetic sex of rana chensinensis by using TRAP molecular marker technology | |
| CN104878099A (en) | Method for detecting single-nucleotide polymorphism of goat ATBF1 gene and application of goat ATBF1 gene | |
| CN105838715B (en) | A kind of microalgae composition type expression promoter and its application | |
| CN104673916A (en) | Method for identifying imprinted gene Rasgrf1 of domestic pig | |
| CN107385078A (en) | Label of pig fat deposition description correlation PNPLA2 mRNA m6A methylate the authentication method and application of function of unit point | |
| CN102191249B (en) | Silkworm Bmlp3 gene promoter and use thereof | |
| CN113186302B (en) | Sex specific molecular marker of Megalobrama amblycephala hybrid Pioneer No. 2 and application | |
| CN101532062A (en) | Amplimer for detecting tibetan sheep cold tolerance gene, detecting agent case and using method thereof | |
| CN110295238A (en) | A kind of relevant OSR1 gene SNP molecular labeling of pteria martensii growth traits and its application | |
| CN112094917B (en) | Western pig blood marginal infiltration site SNP marker related to body length in local pig in China and application | |
| CN111593128B (en) | Molecular marker for identifying genetic sex of northeast wood frog and application method thereof | |
| CN107419026A (en) | Label of pig fat deposition description correlation UCP2 mRNA m6A methylate the authentication method and application of function of unit point | |
| CN103421770B (en) | Heredity markers of pig carcass quality trait and pig meat quality trait related to DIO3 gene and application of heredity markers | |
| CN110982823B (en) | Paralichthys olivaceus oocyte specific gene figla and application thereof | |
| CN110106245B (en) | Method suitable for Erythroculter ilishaeformis gonad tissue mRNA frozen section in-situ hybridization | |
| CN101475990B (en) | PCR-RFLP detection method for complex vertebral malformation of milk cow and crossbred cattle | |
| CN108192984A (en) | Molecular labeling relevant with sheep Meat Quality and specific primer pair and application | |
| CN115851897B (en) | Molecular marker fragment and primer pair for identifying sex of horned beetles and application of molecular marker fragment and primer pair | |
| CN115851977B (en) | Molecular marker and primer pair for identifying sex of pearl soft-shelled turtles and application of molecular marker and primer pair | |
| CN107625969A (en) | The antagonists of MiR 214 are preparing the purposes in being used to mitigate the medicine of renal damage associated conditions caused by albuminuria | |
| CN116042796B (en) | Molecular marker and primer pair for identifying sex of Amyda sinensis and application of molecular marker and primer pair | |
| CN107988386A (en) | A kind of SSR fluorescent dye primers and application for Mandarin fish paternity test |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |