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Method for extracting total ribonucleic acid from plants with polysaccharide and polyphenol by using silica membrane

A technology of silica gel membrane and polysaccharide, which is applied in the field of nucleic acid purification, can solve the problem of unsatisfactory RNA extraction from polysaccharide-rich materials, and achieve the effect of good integrity and high purity

Active Publication Date: 2012-07-04
TIANGEN BIOTECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the RNeasy PlantMini Kit of Qiagen, a well-known foreign biological company, is suitable for most plant materials and has the characteristics of high yield, fast speed, and high purity, but the extraction effect of RNA from polysaccharide-rich materials is not ideal.

Method used

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  • Method for extracting total ribonucleic acid from plants with polysaccharide and polyphenol by using silica membrane
  • Method for extracting total ribonucleic acid from plants with polysaccharide and polyphenol by using silica membrane
  • Method for extracting total ribonucleic acid from plants with polysaccharide and polyphenol by using silica membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Extracting total RNA from white pine needles.

[0042] 1. Add 500 microliters of polysaccharide and polyphenol plant lysate to 100 mg of white pine needles ground by liquid nitrogen, and vortex vigorously to mix;

[0043] 2. Place at room temperature for 5 minutes, and centrifuge the above mixture. The speed of centrifugation is 12000 rpm, the centrifugation time is 2 minutes, and the supernatant of centrifugation is taken out;

[0044]3. Transfer the supernatant in step (2) to a filter column, and perform filtration and centrifugation. The centrifugal filtration time is 1 minute. Carefully draw the supernatant from the collection tube into an RNase-free centrifuge tube, avoiding the tip as much as possible. Contact the cell debris pellet in the collection tube.

[0045] 4. Add 200 microliters of absolute ethanol to the above solution, mix well, transfer to a silica gel membrane adsorption column, and perform adsorption and centrifugation. The speed of centr...

Embodiment 2

[0054] Example 2: Total RNA was extracted from cotton leaves.

[0055] 1. Add 500 microliters of polysaccharide and polyphenol plant lysate to 100 mg of cotton leaves ground by liquid nitrogen, and vortex vigorously to mix;

[0056] 2. Place at room temperature for 5 minutes, and centrifuge the above mixture. The speed of centrifugation is 12000 rpm, the centrifugation time is 2 minutes, and the supernatant of centrifugation is taken out;

[0057] 3. Transfer the supernatant in step (2) into a filter column, and perform filtration and centrifugation. The centrifugal filtration time is 1 minute. Carefully draw the supernatant from the collection tube into a ribonuclease-free centrifuge tube. Avoid touching the cell debris pellet in the collection tube.

[0058] 4. Add 200 microliters of absolute ethanol to the above solution, mix well, transfer to a silica gel membrane adsorption column, and perform adsorption and centrifugation. The speed of centrifugal adsorption is 12000 rp...

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Abstract

The invention relates to a method for extracting total ribonucleic acid from plants with polysaccharide and polyphenol by using a silica membrane, and belongs to the field of nucleic acid purification. The method comprises the following steps of: adding a lysis solution for the plants with polysaccharide and polyphenol into a plant material with polysaccharide and polyphenol, which is ground by liquid nitrogen, performing centrifugal separation on the mixture, taking the supernate obtained through centrifugal separation out, transferring into a filtration column for filtration, adding absolute ethanol into filtrate, uniformly mixing, transferring into a silica membrane adsorbing column for adsorbing centrifugation, adding a protein removing solution and a deoxyribonuclease solution for protein removal centrifugation twice, adding a rinsing solution for desalting centrifugation, performing drying centrifugation, and adding deoxyribonuclease water for rinsing centrifugation to obtain a ribonucleic acid solution. The method has the characteristics of high efficiency, quickness and conciseness; and the purified ribonucleic acid (RNA) can be used for various downstream experiments suchas microarray analysis, in vitro translation, molecular cloning and the like.

Description

technical field [0001] The invention relates to a method for extracting total ribonucleic acid from polysaccharide and polyphenol plants by using a silica gel membrane, belonging to the field of nucleic acid purification. Background technique [0002] The extraction of plant RNA is an important prerequisite for the study of plant molecular biology. RT-PCR, Northern, Real-time PCR, cDNA library construction and other molecular biology researches all require total RNA with high purity and good integrity. Many plants or plant organs contain a large amount of polysaccharides, phenols, and secondary metabolites, or have a high content of endogenous RNase, making it relatively difficult to extract RNA from these plants. The physical and chemical properties of polysaccharides are similar to those of RNA, and they tend to form colloidal precipitates together with RNA during the extraction process, which significantly affects the experimental results in subsequent reverse transcript...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 韩典霖俞萍李晓晨孙克非
Owner TIANGEN BIOTECH BEIJING
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