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Whole-mount in situ hybridization detection method for marine jellyfish polyps

A detection method, in situ hybridization technology, applied in the field of cytogenetics and molecular biology, can solve the problems of overall in situ hybridization detection difficulties, weak probe specificity, complex microstructure, etc., to improve flexibility and operability Sexuality, individual integrity, and strong specificity of hybridization signals

Active Publication Date: 2021-08-31
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Whole-mount in situ hybridization detection method for marine jellyfish polyps
  • Whole-mount in situ hybridization detection method for marine jellyfish polyps
  • Whole-mount in situ hybridization detection method for marine jellyfish polyps

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 seawater PBS (0.1M, pH7.4) Mixture 1 Dilute to 100mL / Mixture 2 80mL Dilute to ...

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 method for detecting whole-body in-situ hybridization of marine jellyfish polyps. The method comprises the following steps: sequentially fixing polyps with paraformaldehyde gradient seawater solution, dehydrating the fixed samples with ethanol solution, hydrating, fixing and acetylating the dehydrated polyps; Pre-hybridization and hybridization; the hybridized samples are subjected to blocking and fluorescent detection. The present invention provides for the first time the whole RNA in situ hybridization step of the marine jellyfish polyp, the jellyfish polyp is individual, the antennae are scattered and unfolded, the jellyfish polyp is clearly colored, the hybridization signal is strong in specificity, and the hybridization background is low. The practical and stable marine jellyfish polyp RNA overall in-situ hybridization technology established by the invention is beneficial to the analysis of gene expression patterns.

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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Patent Type & Authority Patents(China)
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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