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Detection method of marine jellyfish polyps by whole-body in situ hybridization

A detection method and in situ hybridization technology, applied in the field of cytogenetics and molecular biology, can solve the problems of difficult overall in situ hybridization detection, weak probe specificity, complex microstructure, etc., to improve flexibility and operability The effect of high specificity of the hybridization signal, small damage to the antigen, and high specificity

Active Publication Date: 2019-01-15
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Because there are many different shapes and complex microstructures in the developmental stage of the polyp, if the fixation is not good, it will cause the tentacles to shrink and gather, and even detach to varying degrees
[0004] The overall in situ hybridization detection is difficult due to the particularity of the jellyfish's physiological structure, so that the hybridization results obtained by traditional methods have high background signals, weak probe specificity, and unclear coloring.
At present, there is no overall in situ hybridization method for marine jellyfish polyp samples. This patent provides accurate and effective technical support for the study of the function of genes related to marine jellyfish polyp

Method used

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  • Detection method of marine jellyfish polyps by whole-body in situ hybridization
  • Detection method of marine jellyfish polyps by whole-body in situ hybridization
  • Detection method of marine jellyfish polyps by whole-body in situ hybridization

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Step A1, collection of jellyfish polyps

[0056] Take the corrugated board of the jellyfish polyp, put a piece of corrugated board into the filtered seawater, scrape off the polyp for observation, and inhale the polyp at the same developmental stage into the same centrifuge tube. First rinse the polyps with filtered seawater, then rinse with sterilized seawater, and absorb excess sterilized seawater. The polyps in the centrifuge tube were drawn into RNase-free collection tubes in stages, and labeled (date, time, developmental stage). The collection tubes containing the jellyfish polyps were centrifuged quickly and excess sterile seawater was aspirated.

[0057] Step A2, fixation of jellyfish polyps

[0058] Prepare the mixed solution with reference to Table 1 to obtain a clear solution.

[0059] Table 1. Mixed solution preparation table

[0060]

Sterilized sea water

PBS (0.1M, pH7.4)

Mixture 1

Dilute to 100mL

/

Mixture 2

80m...

Embodiment 2

[0086] Step B1: Hydration and digestion of jellyfish polyps

[0087] According to the method described in the above-mentioned Example 1, the jellyfish polyps that were fixed and dehydrated were obtained, and the polyps were transferred into a four-well cell culture plate for hydration, and passed through 70% ethanol aqueous solution, 50% ethanol aqueous solution, and 30% ethanol successively. Rinse with % ethanol NaPBS-Tween solution for 10 min each, and finally rinse with NaPBS-Tween three times, each time for 6 min. Digestion solution was added to each well and left to digest at 25°C for 8 min. To stop the digestion, add stop solution (100mg / mL glycine aqueous solution, the volume ratio of the above digestion solution is 1:50) to each well, quickly absorb it; then dilute 100g / mL glycine aqueous solution with glycine solution (2mg / mL, NaPBS-Tween) Rinse for 5min; finally rinse twice with NaPBS-Tween, 5min / time.

[0088] Step B2: Fixation and acetylation of jellyfish polyps ...

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Abstract

The invention belongs to the technical field of cytogenetics and molecular biology, and relates to a detection method of marine jellyfish polyps by whole-body in situ hybridization. The method comprises the steps as sequentially fixing polymorphic body with paraformaldehyde gradient aqueous solution, dehydrating the fixed sample with ethanol solution, hydrating, fixing and acetylating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, separating the dehydrated polymorphic body, and separating the dehydrated polymorphic body, performing pre-hybridization on the treated sample, and hybridizing. The hybridized samples were sealed and detected by fluorescence colorimetry. The present invention provides an RNA integral in situ hybridization step ofmarine jellyfish polyps for the first time, wherein the jellyfish polyps are intact, the antennae are dispersed and spread, the jellyfish polyps are clearly colored, the hybridization signal specificity is strong, and the hybridization background is low. The invention discloses a practical and stable marine jellyfish polyphasic RNA integral in situ hybridization technology, which is favorable foranalyzing gene expression mode.

Description

technical field [0001] The invention belongs to the technical field of cytogenetics and molecular biology, and relates to an overall in-situ hybridization method for detecting target genes of marine jellyfish polyps. Background technique [0002] Whole mount in situ hybridization is a technique that uses probes such as cRNA or oligonucleotides to detect mRNA expression in intact tissues. This technical method can intuitively reflect the spatiotemporal expression and developmental pattern of the target gene in the development of tissues and organs on the basis of keeping the structure of the tissue or even the organ basically intact, and can be used to analyze the expression of the target gene in the corresponding tissue or organ. role in organ formation. [0003] At present, many breakthroughs have been made in the study of whole-body in situ hybridization of biological tissues and even individuals, and in situ hybridization methods for model animals such as zebrafish and p...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6841
CPCC12Q1/6841C12Q1/6888C12Q2563/107
Inventor 朱玲梁宇君周春娅
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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