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Primers and method for quickly detecting sepiella maindroni embryo apoptosis level

A squid squid and embryo technology, applied in the field of molecular biology, can solve the problems of inability to efficiently evaluate and screen squid squid embryo quality, achieve high accuracy and sensitivity, save time, and improve reproductive efficiency

Active Publication Date: 2020-02-07
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to overcome the problem in the prior art that the quality of squid squid embryos cannot be efficiently evaluated and screened, and provides a primer and method for rapidly detecting the apoptosis level of squid squid embryos

Method used

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  • Primers and method for quickly detecting sepiella maindroni embryo apoptosis level
  • Primers and method for quickly detecting sepiella maindroni embryo apoptosis level

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Extract the RNA of the squid squid embryo to be tested, and use the RNA of the squid squid embryo as a template. The concentration of the obtained total RNA is detected by a nucleic acid quantifier and detected by agarose gel electrophoresis, and then reverse-transcribed to synthesize cDNA , and then using cDNA as a template, use the specific primers related to the Mansoni squid embryo to perform fluorescence quantitative PCR amplification at the marker gene points caspase3 and p53 to obtain amplified fragments; by analyzing the fluorescence quantitative data using The △△Ct method was used to obtain the relative expression of the target gene and evaluate the quality level of squid embryos. Specific steps are as follows:

[0030] 1. Extract the RNA of squid squid embryo;

[0031] The RNA extraction process is to grind the tissue sample, add 0.7mL Trizol solution, and then 2 Ultrasonic cycle stimulation was performed under the ultrasonic wave and then centrifuged.

[0...

Embodiment 2

[0041] The difference with embodiment 1 is:

[0042] 1. Extract the RNA of squid squid embryo;

[0043] The RNA extraction process is to grind the tissue sample, add 0.6mL Trizol solution, and then 2 Ultrasonic cycle stimulation was performed under the ultrasonic wave and then centrifuged.

[0044] 2. Reverse transcription to synthesize cDNA;

[0045] The system for cDNA synthesis by reverse transcription is 5 μl RNase-free Water, 1 μl Anchored Oligo (dT) 18Primer, 1 μl gDNA Remover, 10 μl 2×TS Reaction Mix, 1 μl TransScript RT / RI EnzymeMix, and 1 μl mRNA template.

[0046] 2.1) First add the mRNA template and Anchored Oligo (dT) 18 Primer, mix and centrifuge, heat at 72°C, react for 10 minutes, take it out, and ice-bath for 4 minutes;

[0047] 2.2) Add RNA treatment solution, RNase-free Water, gDNA Remover, 2×TSReaction Mix, TransScript RT / RI Enzyme Mix, etc. to the above system, and place it in a PCR instrument. The condition for reverse transcription to synthesize cDNA i...

Embodiment 3

[0053] The difference with embodiment 1 is:

[0054] 1. Extract the RNA of squid squid embryo;

[0055] The RNA extraction process is to grind the tissue sample, add 0.8mL Trizol solution, and then 2 Ultrasonic cycle stimulation was performed under the ultrasonic wave and then centrifuged.

[0056] 2. Reverse transcription to synthesize cDNA;

[0057] The system for cDNA synthesis by reverse transcription is 6 μl RNase-free Water, 1.5 μl Anchored Oligo (dT) 18Primer, 1.5 μl gDNA Remover, 12 μl 2×TS Reaction Mix, 1.5 μl TransScript RT / RIEnzyme Mix, and 2 μl mRNA template.

[0058] 2.1) First add the mRNA template and Anchored Oligo (dT) 18 Primer, mix and centrifuge, heat at 72°C, react for 10 minutes, take it out, and ice-bath for 4 minutes;

[0059] 2.2) Add RNA treatment solution, RNase-free Water, gDNA Remover, 2×TSReaction Mix, TransScript RT / RI Enzyme Mix, etc. to the above system, and place it in a PCR instrument. The reverse transcription synthesis cDNA condition is ...

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Abstract

The invention relates to the field of molecular biology, and discloses primers and method for quickly detecting the sepiella maindroni embryo apoptosis level to solve the problem that in the prior art, the sepiella maindroni embryo quality cannot be efficiently evaluated and screened. The method comprises the preparation steps: (1) RNA of a sepiella maindroni embryo is extracted; (2) cDNA is synthesized through reverse transcription; (3) the cDNA is taken as a template, real-time fluorescent quantitative PCR amplification is conducted through a caspase3 primer sequence and a p53 primer sequence, and the relative expression levels of caspase3 and p53 genes are obtained through amplification. The invention discloses the detection method and primer sequences suitable for the sepiella maindroni embryo apoptosis level for the first time, the technical blank of quick detection of the quality of cephalopod oosperm is filled, the quality situations of chromosomes and cell nucleuses of embryoscan be timely and accurately obtained, an effective method is provided for quality change of the cephalopod embryos, and the breeding efficiency of sepiella maindroni is improved greatly.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer and a method for rapidly detecting the apoptosis level of squid squid embryos. Background technique [0002] Apoptosis is a programmed death process in which the cell death pathway is activated under the induction of specific endogenous and exogenous signals and occurs under the regulation of related genes. It is known that apoptosis is the only way in the process of biological development, such as the metamorphosis of tadpoles, the metamorphosis of insects, the disappearance of interdigital webs during the embryonic development of higher mammals, the formation of eye vitreous and lens, etc. A large number of research results have shown that the disorder of apoptosis process in the process of embryonic development will directly lead to the blockage of embryonic development and cause embryonic death. At present, there is still a lack of rapid detection methods for the ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2531/113C12Q2561/113C12Q2563/107Y02A40/81
Inventor 柳意樊刘炳舰孟方黄友坤王起
Owner ZHEJIANG OCEAN UNIV
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