Method and kit for extracting ribonucleic acid (RNA)

A kit and extraction aid technology, applied in the field of molecular biology, can solve the problems of cumbersome operation, cumbersome operation steps, RNA failure, etc., and achieve the effect of wide application range and simple and fast method

Active Publication Date: 2011-01-05
中生方政生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic method of RNA extraction, the TRIZOL method, is a method based on the principle of guanidine isothiocyanate / phenol / chloroform extraction. This method has a wide range of applications, and is suitable for both animals and plants. The operation steps of the method are cumbersome, and toxic reagents such as phenol and chloroform are used in the extraction reagents, and this method is not suitable for the extraction of RNA from materials rich in polysaccharides and polyphenols
The RNeasy Plant Mini Kit of Qiagen, a well-known foreign biological company, is a kit specially designed for RNA extraction from plant materials. The method used in the kit is based on the principle of cleavage of guanidine isothiocyanate. Applicable, with the advantages of fast extraction speed, high purity, and no use of toxic reagents, etc., but the RNA extraction effect is not ideal for animal materials and microbial materials, especially materials rich in polysaccharides. Since polysaccharides and RNA are not easy to separate, it is easy to put the adsorption column clogging, resulting in failure of RNA extraction
The CTAB-LiCl method is often used to extract RNA from polysaccharide-rich polyphenol materials, but since CTAB itself is a reagent similar in function to SDS, although it can form insoluble complexes with RNA and DNA, it is beneficial to remove polysaccharides , but the CTAB method is usually combined with other operations, such as LiCl precipitation and other steps, making the method cumbersome to operate, and the toxic reagent chloroform is used in the extraction

Method used

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  • Method and kit for extracting ribonucleic acid (RNA)
  • Method and kit for extracting ribonucleic acid (RNA)
  • Method and kit for extracting ribonucleic acid (RNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: the kit for extracting RNA according to the present invention

[0067] The kit for extracting RNA of the present invention includes a lysate, a protein-removing solution, and a washing solution.

[0068] The lysate contained 3M guanidine isothiocyanate, 10 mM Tris-HCl with a pH value of 6.5, and 1% β-mercaptoethanol. The specific preparation method is: weigh 35.45g of guanidine isothiocyanate, add enough RNase-free distilled water to fully dissolve, then add 1.0ml of Tris-HCl with a pH of 6.5 and a concentration of 1M, mix well, and dilute to 99ml , add 1 ml of β-mercaptoethanol.

[0069] The protein-removing solution contains 0.1M guanidine isothiocyanate, 5mM Tris-HCl with a pH value of 7.5, and 5% absolute ethanol. Weigh 1.18g of guanidine isothiocyanate, add RNase-free distilled water to fully dissolve, then add 0.5ml of Tris-HCl with a pH of 6.5 and a concentration of 1M, and finally add 10ml of RNase-free absolute ethanol, and set the volume to 100...

Embodiment 2

[0071] Embodiment 2: the kit for extracting RNA of the present invention

[0072] The kit for extracting RNA of the present invention comprises a lysate, a protein-removing solution, a rinsing solution, an eluent and a glass cellulose membrane. Among them, the lysate contains 5M guanidine isothiocyanate, 50mM Tris-HCl with a pH value of 8.0, and 1% β-mercaptoethanol; the deproteinized solution contains 0.5M guanidine isothiocyanate, 50mM Tris-HCl, 20% absolute ethanol; the rinse solution contains 20 mM Tris-HCl with a pH value of 8.0, 200 mM NaCl, and 80% absolute ethanol; the eluent is 0.1% DEPC with a pH value of 8.0 sterilization Treat water; glass cellulose membrane is 0.45 μm, Whatman company, GF / B type. The configuration method is the same as that in Embodiment 1.

Embodiment 3

[0073] Embodiment 3: the kit for extracting RNA according to the present invention

[0074] The kit for extracting RNA of the present invention comprises a lysate, a protein-removing solution, a rinsing solution, an eluent and a glass cellulose membrane adsorption column. Among them, the lysate contains 4M guanidine isothiocyanate, 25mM Tris-HCl with a pH value of 7.5, and 1% β-mercaptoethanol; the deproteinized solution contains 0.3M guanidine isothiocyanate, 10mM Tris-HCl with a pH value of 7.5 -HCl, 10% absolute ethanol; the rinsing liquid contains 10 mM Tris-HCl with a pH value of 7.5, 100 mM NaCl, and 80% absolute ethanol; the eluent is sterile distilled water with a pH value of 7.5 without nuclease; The glass cellulose membrane adsorption column is a 0.45 μm glass cellulose membrane, Whatman Company, GF / B type, 1.5 mL spin column. The configuration method is the same as that in Embodiment 1.

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Abstract

The invention relates to the field of molecular biology and discloses a method and a kit for extracting ribonucleic acid (RNA). The method for extracting the RNA comprises the following steps of: uniformly mixing a sample and lysis solution; centrifugally collecting supernate and uniformly mixing the supernate and absolute ethyl alcohol which is half of the volume of the supernate; combining the mixture with 0.45 mu m glass fiber cellulose acetate membrane; washing with deproteinization liquid and rinsing liquid; eluting adsorbed RNA; uniformly mixing lysis cells and an extraction aid; and centrifugally removing polysaccharide and polyphenol substances. The kit for extracting the RNA comprises the lysis solution, the deproteinization liquid, the rinsing liquid, the eluting liquid and the extraction aid. The method of the invention has the advantages of simpleness, rapidness, no use of toxic reagent, wide application range and capabilities of effectively removing the polysaccharide andpolyphenol substances and separating high-quality RNA out by adding the extraction aid. The kit of the invention has the advantages of no toxic reagent, wide application range, RNA extraction effectsof plant materials which are rich in polysaccharide and polyphenol superior to that of foreign kits, low cost and suitability for extensive laboratories and scientific researches.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method and kit for extracting RNA. Background technique [0002] Extracting RNA from tissues is a necessary prerequisite for molecular biology research. Extracting RNA with high purity and integrity is useful for Northern hybridization analysis, mRNA isolation, in vitro translation, cDNA library construction, RT-PCR gene isolation, and differential display analysis. researches are of great significance. Because many materials are rich in phenolic compounds and polysaccharides and contain some unidentified secondary metabolites, it is extremely difficult to effectively isolate and purify RNA from these materials, thus hindering the progress of their molecular biology research. [0003] Commonly used RNA extraction methods include TRIZOL method, guanidine isothiocyanate method, CTAB-LiCl method, and total RNA extraction kits from domestic and foreign biological companies. The c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李艳萍
Owner 中生方政生物技术股份有限公司
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