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Method for extracting total RNA from hemolymph of procambarus clarkii

A technology of Procambarus clarkii and hemolymph, which is applied in the field of extracting total RNA of Procambarus clarkii hemolymph, can solve problems such as individual death of Procambarus clarkii, amplification differences, unfavorable genes, etc., and achieve stable extraction effect and good quality , the effect of easy operation

Inactive Publication Date: 2021-03-09
HUAZHONG AGRI UNIV
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Problems solved by technology

However, due to the large amount of hemocyanin and other interfering factors in the hemolymph, there is no stable and efficient method for extracting RNA from the hemolymph.
In addition, the existing methods for extracting RNA from other tissues will lead to the death of Procambarus clarkii individuals. In terms of experimental design, we can only use the same treatment method of different individuals to study the continuous changes in the body. This experimental method It magnifies the differences between individuals, which is very unfavorable for studying the continuous changes of genes in the same individual under conditions such as exogenous stress

Method used

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  • Method for extracting total RNA from hemolymph of procambarus clarkii
  • Method for extracting total RNA from hemolymph of procambarus clarkii
  • Method for extracting total RNA from hemolymph of procambarus clarkii

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Embodiment 1

[0031] A method for extracting Procambarus clarkii hemolymph RNA, comprising the following steps:

[0032] Step A: Hemolymph Aspiration and Isolation of Blood Cells

[0033] (1) Take 390-400 μl (about 400 μl) blood sample from the shrimp body with a 1ml syringe, mix it with anticoagulant at a volume ratio of 1:1 to 400 μl, place it in an EP tube, and place it on ice;

[0034] (2) Centrifuge at 800×g, 4°C for 20 minutes to separate blood cells;

[0035] (3) Discard the supernatant, keep the white precipitate, add 200 μl of pre-cooled Trizol reagent, use a grinder to grind until the white precipitate disappears, and make the Trizol solution pink, and add 800 μl of Trizol reagent to each sample;

[0036] (4) Stand at room temperature for 5 minutes, then centrifuge at 12000×g for 10 minutes at 4°C;

[0037] (5) Take 900 μl of the supernatant and place it in a new EP tube, do not inhale the precipitate; freeze the collected liquid at -80°C or directly use it for RNA extraction; ...

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Abstract

The invention belongs to the technical field of preparation technology of RNA samples from aquatic animals, and specifically relates to a method for extracting total RNA from hemolymph of procambarusclarkii. The method includes two main steps of extracting the hemolymph of procambarus clarkii and separating blood cells and extracting the total RNA from the hemolymph samples. The extraction of thetotal RNA can be completed with only a small amount of hemolymph, so that the procambarus clarkii can be still alive after blood drawing, and the observation and study on the genetic changes in the bodies of the procambarus clarkii can be continuously carried out. The extracted total RNA is excellent in quality and little in degradation and can be used for high-throughput sequencing; and the method is low in cost, simple in operation and stable in extraction effect, and can guarantee the activity of the procambarus clarkii while extracting the total RNA from the hemolymph.

Description

technical field [0001] The invention belongs to the technical field of aquatic animal RNA sample preparation, and in particular relates to a method for extracting total RNA from the hemolymph of Procambarus clarkii. Background technique [0002] Procambarus clarkii (Procambarus clarkii), also known as red swamp crayfish, is called "crayfish" in our country. Belonging to the phylum Arthropoda, Crustacea, Decapod order, Crawfish family, Procambarus genus. Native to northern Mexico and the southern United States, it was first transplanted in the Honshu region of Japan in 1918. Introduced into Nanjing, my country in 1929, due to its extremely strong environmental adaptability, reproductive ability, and strong dissemination power, it has now spread all over the Yangtze River Basin in China and has become an important economic aquatic resource in my country. At present, the research on Procambarus clarkii is also deepening day by day, gradually improving and understanding the re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806
Inventor 白旭峰任鑫
Owner HUAZHONG AGRI UNIV
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