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Method for rapidly extracting RNA from animal tissue

An animal tissue and extraction method technology, applied in the field of molecular biology, can solve the problems of inability to obtain an RNA extraction method, high toxicity of reagents, complicated operation, etc., and achieve the effects of considerable market prospect, low reagent cost and simple operation.

Inactive Publication Date: 2018-06-05
上海怡泽生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experimental results show that this method is not effective in extracting RNA from tissues; the disadvantage of TRIzol method is that it takes a long time, the operation is complicated, and the reagents are highly toxic.
Therefore, in the case of failure to simply use the existing kits, the applicant of the present invention deeply studied the principles of various existing tissue RNA extraction methods (mainly the TRIzol method), and analyzed the role of each step in the RNA extraction , which proves that based on the working principle of TRIzol method, a faster RNA extraction method cannot be obtained

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation of cells from mouse liver tissue

[0037] 1. After dissection, cut the mouse liver tissue into pieces the size of half a mung bean (with a weight between 10 and 50 mg), put them into a weighed and marked centrifuge tube, and weigh them.

[0038] 2. According to the ratio of 100 μl PBS per 10 mg of tissue, add pre-cooled PBS to the centrifuge tube containing the tissue block, and grind it evenly on ice. Grinding time should be controlled within 2 minutes as much as possible.

[0039] 3. Take a new centrifuge tube, add 100 μl tissue homogenate, and centrifuge at 300 rpm for 1 minute.

[0040] 4. Take 10 μl supernatant to a new centrifuge tube, add 90 μl PBS to mix, and centrifuge at 1500 rpm for 3 minutes.

[0041] 5. Carefully remove the supernatant, the pellet is the isolated cells, and set aside.

Embodiment 2

[0042] Embodiment two: the preparation of nucleic acid extraction reagent

[0043] According to the method proposed by the applicant of the present invention (patent name "A method and reagent for extracting nucleic acid from cells", patent application number 2016108773755), the nucleic acid extraction reagent was prepared. The formula is as follows:

[0044] 1% Triton X-100;

[0045] 1U / μL mouse RNase inhibitor;

[0046] 10mmol / L DTT;

[0047] 1mmol / L EDTA;

[0048] 10mmol / L Tris-HCl, pH8.0.

Embodiment 3

[0049] Example three: RNA extraction from isolated cells

[0050] 1. Add 100 μl of nucleic acid extraction reagent (see Example 2 for the formula) to the cell pellet obtained according to the method described in Example 1, and repeatedly blow and suck several times with a pipette to fully suspend the cells and disperse the cells evenly.

[0051] 2. Stand at room temperature for 10 minutes to fully lyse the cells and release the nucleic acid into the solution.

[0052] Heat at 3.75°C for 10 minutes to inactivate RNases.

[0053] 4. Place on ice to allow the solution to cool. This solution can be directly used for gene expression level detection or gene cloning and other operations, and can also be stored at -20°C or -80°C for subsequent experiments.

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Abstract

The present invention relates to the field of molecular biology, and particularly to a method for rapidly extracting RNA from animal tissues. The method mainly comprises two steps such as rapid separation of cells from tissue and rapid extraction RNA from cells with a nucleic acid extraction reagent, wherein the step 1 comprises: cutting tissue into small blocks, weighing, adding PBS, uniformly grinding to obtain tissue homogenate, carrying out low-speed centrifugation to precipitate large tissue residue so as to make the single cell suspend in the supernatant, taking the supernatant with thesuspended cells, adding PBS, uniformly mixing, and carrying out centrifugation to remove the supernatant to obtain precipitate, ie., the separated cells, and the step 2 comprises: adding a nucleic acid extraction reagent to the cells, uniformly mixing, carrying out room temperature standing to lysing the cells, heating for 10 min at a temperature of 75 DEG C, and cooling the sample by placing on ice so as to obtain the prepared RNA for gene expression level detection or gene cloning and other works. According to the present invention, the method has advantages of simple operation, high speed,low reagent cost, no toxicity and no odor.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for quickly extracting RNA from animal tissues. Background technique [0002] Before biomedical research and clinical work such as gene expression level detection or gene cloning, nucleic acid must be extracted from biological samples. Biological samples mentioned here include cultured cells, animal and plant tissues, body fluids (such as blood, sputum, interstitial fluid, etc.), microbial samples, etc. As we all know, nucleic acids include DNA and RNA. DNA is the carrier of genetic information, while RNA is an intermediate bridge that transforms the genetic information carried by DNA into functional proteins; in addition, RNA also has a variety of complex regulatory functions in organisms. When it is necessary to detect gene expression levels to study related biological functions, RNA is an extremely important and commonly used research object. The first step in dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2523/113C12Q2527/127
Inventor 刘晓雷袁新旺管延斌薛勰
Owner 上海怡泽生物科技有限公司
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