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Method suitable for in-situ hybridization of cherax quadricariratus gonadal tissue mRNA paraffin section

A technology of in situ hybridization and red-clawed crayfish, applied in the direction of DNA / RNA fragments, recombinant DNA technology, DNA preparation, etc., can solve the problems of unclear mechanism of shrimp sexual phenotype differentiation and dimorphic development, and achieve tissue The shape is kept intact, the nucleoplasm is distinct, and the design is reasonable

Active Publication Date: 2019-07-09
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of sex phenotype differentiation and dimorphic development in this shrimp is still unclear

Method used

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  • Method suitable for in-situ hybridization of cherax quadricariratus gonadal tissue mRNA paraffin section
  • Method suitable for in-situ hybridization of cherax quadricariratus gonadal tissue mRNA paraffin section
  • Method suitable for in-situ hybridization of cherax quadricariratus gonadal tissue mRNA paraffin section

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A method suitable for probes for in situ hybridization of gonad tissue mRNA paraffin sections of red-clawed crayfish, according to the following steps:

[0039] Step S1: Design and construct the primer sequence of the dsx probe vector according to the sequence of the coding region of the cichlid dsx gene, the forward and reverse primers respectively have EcoRI and Xhol restriction sites, as follows:

[0040] The forward primer is: 5'-GAATTCTAATACGACTCACTATAGGGA-3',

[0041] The reverse primer is: 5'-CCTATAGTGAGTCGTATTAAAGCTT-3';

[0042] Step S2: Using the cDNA of the gonad tissue of the red crayfish obtained by reverse transcription as a template, use primers to amplify a part of the ORF sequence of the dsx gene by PCR reaction, which is used as a probe sequence specific to the dsx protein, and the PCR method is 94°C for 4 minutes ; 94°C for 30s, 55°C for 30s, 72°C for 1min, 32cycle; 72°C for 7min, use the Axygen Gel Recovery Kit to purify the target fragment;

[004...

Embodiment 2

[0057] A method suitable for in situ hybridization of gonad tissue mRNA paraffin sections of redclaw crayfish, comprising gonad tissue embedding, paraffin sections, in situ hybridization and photographing records, the in situ hybridization adopts the above-mentioned probes.

[0058] The above-mentioned in situ hybridization comprises the following steps:

[0059] 1) Tissue fixation: After the tissue is taken out and washed, it is immediately placed in fixative solution (prepared with DEPC water) and fixed for 2-12 hours;

[0060] 2) Dehydration: After the tissue is fixed, it is dehydrated by graded alcohol, soaked in wax, and embedded;

[0061] 3) Slicing: Paraffin wax sliced ​​by a slicer, picked up by a spreader, and baked in a 62°C oven for 2 hours;

[0062] 4) Paraffin sections were dewaxed to water: put the sections into xylene Ⅰ 15 min-xylene Ⅱ 15 min-absolute ethanol Ⅰ 5 min-absolute ethanol Ⅱ 5 min-85% alcohol 5 min-75% alcohol 5 min-DEPC washing;

[0063] 5) Digesti...

Embodiment 3

[0074] In order to improve the dewaxing effect of paraffin sections, the further optimization scheme is as follows:

[0075] Paraffin sections were dewaxed to water step containing 0.03-0.06% chloral and 2.8-3.0% lumefantrine in xylene. On the one hand, the presence of chloral and lumefantrine promotes the rapid dissolution of paraffin used to support tissues and cells in toluene, avoiding tissue shrinkage and cell deformation, so that the tissue remains in place and the tissue shape remains intact; on the other hand, it can Increase the permeability of the antibody to the cell membrane. During the process of DAB color development and hematoxylin staining, the cells are well colored, the nucleoplasm is distinct, the structure is clear, the tissue and cells are more transparent and clear, and it is easy to observe, improving the efficiency of in situ hybridization. Accuracy; In addition, it can also increase the toughness of the DNA chain, avoid the formation of complexes betwe...

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Abstract

The invention provides a method suitable for in-situ hybridization of a cherax quadricariratus gonadal tissue mRNA paraffin section, and belongs to the technical field of in-situ hybridization. The method comprises the steps of gonadal tissue embedding, paraffin sectioning, in-situ hybridization, and photographing and recording. The sequence of a probe used in in-situ hybridization is as shown inSEQ ID NO.3. The probe has clear specificity on tissue positioning of a dsx gene, so that the display effect on the mRNA level of the dsx gene is realized. The method for mRNA in-situ hybridization ofthe paraffin section is established in cherax quadricariratus to research expression modes of related genes in the gonad, and the expression location of the sex-related genes of the cherax quadricariratus in the gonad can be clearly described. The method avoids tissue shrinkage and cell deformation while the dewaxing effect of the paraffin section is good, so that the tissue is still kept in theoriginal position, the tissue morphology is kept complete, the enzymolysis effect can be improved, cell coloring is good, nucleoplasm is clear, and the in-situ hybridization accuracy is finally improved.

Description

technical field [0001] The invention belongs to the technical field of in-situ hybridization, and in particular relates to a method suitable for in-situ hybridization of mRNA paraffin sections of gonad tissue of red-clawed crayfish. Background technique [0002] Red-clawed crayfish (Cherax quadricariratus), commonly known as Australian freshwater lobster, is native to tropical northern Australia and southern New Guinea. One of the world's rare freshwater economical shrimps. Like other freshwater decapods, redclaw males grow faster and are larger than females. The red-clawed squid has been widely cultured, especially in the past ten years, because of its obvious biological characteristics, the aquaculture of the red-clawed squid has developed very fast. However, the mechanism of sexual phenotype differentiation and dimorphic development in this shrimp is still unclear. In situ hybridization tissue (or cell) chemistry (In situ Hybridization Histochemistry, ISHH) referred to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6811C12Q1/6841C12N15/11C12N15/10
CPCC12Q1/6888C12Q1/6811C12Q1/6841C12Q2600/158Y02A40/81
Inventor 郑建波贾永义顾志敏程顺李飞迟美丽刘士力蒋文枰
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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