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RNA extraction method by using silicon film pole to adsorb RNA

An extraction method and column adsorption technology, which is applied in the field of RNA extraction using silicon membrane column to absorb RNA, can solve the problems of DNA pollution, shorten the extraction time, etc., and achieve the effect of simple formula, shortened extraction time and high purity

Inactive Publication Date: 2009-02-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a simple, effective and rapid RNA extraction method that utilizes a silicon membrane column to absorb RNA, solves the DNA pollution phenomenon in the existing RNA extraction technology, shortens the extraction time, and the extraction process is easy automation

Method used

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  • RNA extraction method by using silicon film pole to adsorb RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Preparation of denaturing solution:

[0058] ①Take an 80ml bottle, weigh 3.65g of solid guanidine isothiocyanate, and put it into the bottle;

[0059] ②Weigh 1.54g sodium citrate and put it into the bottle;

[0060] ③ Dilute to 10ml with double distilled water;

[0061] ④Heat in a water bath at 60°C until completely dissolved, then adjust the pH to 4.0 with citric acid;

[0062] ⑤Store at room temperature for later use.

[0063] Steps to extract RNA:

[0064]①Add 1000 μl denaturing solution and 240 μl chloroform to the prepared Pseudomonas strain M18, vortex and mix well, centrifuge at 12,000 rpm at 4°C for 1 min, and the bacterial solution is divided into three layers in the tube;

[0065] ② Transfer 500 μl of the upper liquid to a silicon membrane column, add 250 μl of isopropanol, and centrifuge at 12,000 rpm for 1 min;

[0066] ③ Add 500 μl of 75% ethanol washing solution to the silicon membrane column, and centrifuge at 12,000 rpm for 1 min;

[0067] ④Repeat ...

Embodiment 2

[0073] Preparation of denaturing solution:

[0074] The steps are the same as in Example 1, wherein: 3.65g sodium lauryl sarcosine, 2.95g sodium acetate, dilute to 10ml with double distilled water, heat in a 60°C water bath until completely dissolved, then adjust the pH to 4.0 with pure acetic acid. Set aside at room temperature.

[0075] Steps to extract RNA:

[0076] ①Add 1000 μl denaturing solution and 240 μl chloroform to the prepared yeast cells, vortex and mix well, centrifuge at 12,000 rpm at 4°C for 1 min, and the bacterial solution is divided into three layers in the tube;

[0077] ② Transfer 500 μl of the upper layer liquid to a silicon membrane column, and centrifuge at 12,000 rpm for 1 min;

[0078] ③ Add 500 μl of 75% ethanol washing solution to the silicon membrane column, and centrifuge at 12,000 rpm for 1 min;

[0079] ④Repeat the previous step;

[0080] ⑤Centrifuge at 12,000rpm for 1min to ensure that there is no liquid in the silicon membrane column;

[...

Embodiment 3

[0085] Preparation of denaturing solution:

[0086] The procedure is the same as in Example 1, wherein: 2ml of saturated phenol, 3.05g of sodium lactate, and distilled water to 10ml. Heat in a water bath at 60°C until completely dissolved, then adjust the pH to 4.0 with lactic acid. Set aside at room temperature.

[0087] Steps to extract RNA:

[0088] ① Add 1000 μl denaturing solution and 240 μl chloroform to the prepared fresh tea sample, vortex and mix well, centrifuge at 12,000 rpm at 4°C for 1 min, and the bacterial solution is divided into three layers in the tube;

[0089] ② Transfer 500 μl of the upper liquid to a silicon membrane column, add 250 μl of isopropanol, and centrifuge at 12,000 rpm for 1 min;

[0090] ③ Add 500 μl of 75% ethanol washing solution to the silicon membrane column, and centrifuge at 12,000 rpm for 1 min;

[0091] ④Repeat the previous step;

[0092] ⑤Centrifuge at 12,000rpm for 1min to ensure that there is no liquid in the silicon membrane c...

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Abstract

The present invention relates to a simple, effective and quick RNA extraction method for absorbing RNA by utilizing a silicon film column. The method includes the following steps: after cells are collected, denaturing solution and chloroform are added into the cells, uniformly mixed and centrifugated until the bacteria solution is divided into three layers in a tube; the top layer of liquid is extracted and transferred into the silicon film column to be centrifugated; the previous step is repeated; centrifugation is conducted again in order to ensure that no liquid exists in the silicon film column; double distilled water and eluent are added into the silicon film column; and after centrifugation, the eluent is collected into a clean centrifugal tube. The denaturing solution contains split agent, RNA extraction agent and water; wherein, the split agent is guanidinium isothiocyanate, sodium dodecyl sarcosinate or phenol; and the RNA extraction agent is sodium citrate, sodium acetate, sodium lactate, ascorbic acid, potassium malate, sodium oxalate or potassium tartrate. The extraction time of the method is not longer than 6 minutes, DNA pollution cannot be generated, the extraction process can be easily automated, and the purity of the extracted RNA is high.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a method for extracting RNA by using a silicon membrane column to absorb RNA. Background technique [0002] At present, many studies need to quickly extract RNA from cells or tissues. There are various methods for extracting RNA at home and abroad, such as "The single-step method of RNA" published in "Nat Protoc" ("Nature") 2006, 1 (2): 581-585 by American scientist Chomczynski P, etc. isolation by acidguanidinium thiocyanate-phenol-chloroform extraction[J]” (“Using acid phenol-guanidinium isothiocyanate-chloroform to extract RNA in one step[J]”), this paper proposed a kind of acid phenol-guanidine isothiocyanate - Chloroform method. At present, the acid phenol-guanidine isothiocyanate-chloroform method is considered to be the most standard method at home and abroad. The basic principle is to use guanidine isothiocyanate to lyse cells. Guanidine isothiocyanat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02C12N15/10
Inventor 董德贤何静高倩卢奕李荣秀
Owner SHANGHAI JIAO TONG UNIV
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