Method and kit for extracting mRNA (massager ribonucleic acid) from fungi rich in polysaccharides

A polysaccharide and fungus technology, applied in the field of nucleic acid purification, can solve the problems of high price and high extraction cost, and achieve the effect of reducing the extraction cost

Inactive Publication Date: 2016-12-07
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this technical solution cannot be directly applied to polysaccharide-rich fungi...

Method used

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  • Method and kit for extracting mRNA (massager ribonucleic acid) from fungi rich in polysaccharides

Examples

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Effect test

Embodiment 1

[0084] The mRNA was extracted from polysaccharide-rich Rhizoctonia graminearum by the method described above.

[0085] Some purchased test materials are as follows:

[0086] 2ml spin column, purchased from Shenzhen Comma Biotechnology Co., Ltd., catalog number 007400. The thickness of the sieve plate is 1.6mm, and the aperture is 20μm.

[0087] Cellulose powder, purchased from Sigma Company, product number C6288, CAS number 9004-34-6.

[0088] The specific extraction process is:

[0089] (1) Weigh 0.1 g of cellulose powder, add it to a spin column, add 700 μl of washing buffer to soak for 2 hours, centrifuge, and discard the filtrate. Add 700 μl of washing buffer, centrifuge, discard the filtrate, and set aside the spin column.

[0090] (2) Scrape 0.2 g of fresh mycelium from a plate covered with cellophane, add liquid nitrogen in a mortar, and grind it into powder. Transfer to a centrifuge tube, add 650 μl CTAB extract (3.5 μl proteinase K (20 mg / ml) before use, final co...

Embodiment 2

[0103] This embodiment is a kit for extracting mRNA from polysaccharide-rich fungi, which consists of the following parts:

[0104] The spin column has a sieve plate with a preset aperture in the spin column. The sieve plate divides the interior of the spin column into the upper space for containing the solid-liquid mixture or the solid phase left after centrifugation, and for discharging the centrifuged through the sieve. The lower space of the liquid phase of the plate;

[0105] Centrifuge tube;

[0106] cellulose powder;

[0107] The mixed solution is formed by mixing phenol: chloroform: isoamyl alcohol = volume ratio 25±5:24±5:1; absolute ethanol; ethanol with a volume ratio of 75±5%; DEPC-treated H 2 O; Isopropanol; Proteinase K solution;

[0108] CTAB extract; wash buffer; elution buffer;

[0109] The washing buffer is STE buffer containing 30-35% ethanol by volume; the elution buffer is STE buffer containing 17-20% ethanol by volume; the washing buffer and elution b...

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Abstract

The invention relates to a method and kit for extracting mRNA (massager ribonucleic acid) from fungi rich in polysaccharides. The method comprises steps as follows: a spin column is prefabricated with cellulose powder; impurities in fungal hyphae are removed with a CTAB (cetyltrimethyl ammonium bromide) extracting solution and an organic solvent mixed liquid; mRNA is absorbed to the cellulose powder, the mRNA is eluted and precipitated after cleaning and impurity removal, and precipitates are dissolved and stored for standby application. The kit comprises the spin column, a centrifugal tube, the cellulose powder, the CTAB extracting solution, a cleaning buffer solution, an eluting buffer solution and the like. According to the method and the kit, the polysaccharose substances in the fungi can be effectively removed, high-quality mRNA is obtained, and meanwhile, the extraction cost can be remarkably reduced.

Description

technical field [0001] The invention relates to a method for extracting mRNA from polysaccharide-rich fungi and a kit thereof, belonging to the technical field of nucleic acid purification. Background technique [0002] As far as the applicant knows, there are currently two main methods for extracting and purifying fungal mRNA at home and abroad: one is the one-step extraction method of guanidine isothiocyanate (Trizol). The method of separating the organic phase and the aqueous phase with chloroform removes protein, DNA, etc., and finally the mRNA is precipitated. The other is the silica gel membrane adsorption extraction method. The broken fungal hyphae are extracted with phenol and chloroform to remove protein, then the silica gel membrane is used to absorb nucleic acid, and different eluents are used to remove DNA, and finally eluted to obtain mRNA. [0003] However, when extracting mRNA from filamentous fungi that contain more polysaccharides, because the polysaccharid...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 李伟陈怀谷孙海燕邓渊钰张爱香
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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