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Method for extracting mycotic spore RNA by mechanical disruption method

A mold spore and mechanical crushing technology, which is applied in the field of extracting mold spore RNA by mechanical crushing method, can solve the problem that the method of single-cell sequencing cannot be applied to mold spores, etc., and achieve good reference value, convenient operation and control, and high extraction efficiency.

Active Publication Date: 2017-11-21
CHINA AGRI UNIV
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Problems solved by technology

Traditional methods require a large number of samples, making single-cell sequencing methods unsuitable for the study of mold spores

Method used

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  • Method for extracting mycotic spore RNA by mechanical disruption method
  • Method for extracting mycotic spore RNA by mechanical disruption method
  • Method for extracting mycotic spore RNA by mechanical disruption method

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Experimental program
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Effect test

Embodiment

[0048] 1. Experimental materials

[0049] The mold spores used in the following experiments are mainly Aspergillus niger spores. The Aspergillus niger strains were purchased from Shanghai Fuxiang Biological Company and stored in 0.9% sterile sodium chloride solution containing 15% glycerol. The freeze-dried strains were used as generation 0 , mainly the 3rd and 4th generations in the experiment. Cultivated according to conventional methods in the art.

[0050] 2. The mold spore mechanical crushing method used in the following experiments: take the mold spore liquid, add glass beads (hereinafter referred to as beads), carry out mechanical crushing (grinding) treatment, and then perform RNA extraction.

[0051] 3. The RNA extraction method used in the following experiments:

[0052] The mold spore liquid after the mechanical crushing (grinding) treatment is directly subjected to RNA extraction.

[0053] The specific process of RNA extraction is as follows:

[0054] (1) Get t...

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Abstract

The invention relates to a method for extracting mycotic spore RNA by a mechanical disruption method. The method includes steps of taking mycotic spore liquid, adding glass bead to perform mechanical disruption treatment, and then performing RNA extraction. In the invention, a mechanical method is applied to break a mycotic spore wall, and RNA extraction and purification are carried out by combining with a column purification method after breaking; then reverse transcription is performed by using a kit, and the extracting effect is detected through a real-time fluorescent quantitation PCR and gel electrophoresis; accordingly, a technical system for accurately extracting the mycotic spore RNA is set up. The optimized method for extracting mycotic spore RNA has the advantages of being high in extracting efficiency, convenient and easy to operate, free from toxicity and harm; more importantly, the extracted RNA quality is good, and the RNA can meet the past sequencing requirement of monoplast; the method provides a good reference value for the monospore sequencing in future.

Description

technical field [0001] The invention relates to a method for extracting mold spore RNA by a mechanical crushing method. Background technique [0002] Although there are many methods for RNA extraction, many of which have matured, but for fungi with complex cell wall components, these common nucleic acid extraction methods have limitations. Fungi are rich in polysaccharides, pigments, nucleases and other substances that make RNA extraction difficult. It is more difficult to isolate and isolate, especially the RNA extraction of mold spores that contain a harder spore wall is more limited. [0003] On the other hand, due to the emergence of single-cell whole-genome sequencing technology, our traditional large-sample cell sequencing method has been changed, and it has also changed from the analysis of a cell population to the analysis of a single cell, which can provide us with more valuable For example, in different locations of mold culture dishes, the gene composition of mol...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12R1/685
CPCC12N15/1003
Inventor 罗云波许文涛黄昆仑田兰芳贺晓云郭明璋
Owner CHINA AGRI UNIV
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