Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols

A technology of plant materials and polyphenols, which is applied in the field of RNA extraction, can solve the problems of the measurement of interference RNA ultraviolet absorption, etc., and achieve the effect of simple operation, high concentration and short extraction time

Inactive Publication Date: 2015-02-18
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When Tesniere et al. used the phenol method and the guanidine method to extract the RNA of grape fruit, they found that the RNA condensed with an unknown compound to form an insoluble complex, and this unknown compound had strong light absorption at 230nm and 270nm, thereby interfering with the RNA Determination of UV absorption

Method used

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  • Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols
  • Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols
  • Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Extraction of Phalaenopsis leaf total RNA

[0035](1) Take 1 g of fresh Phalaenopsis leaves, put them into a mortar pre-cooled by liquid nitrogen, add liquid nitrogen and grind them fully until they are powdery, add 1000 μL of extraction buffer A immediately, vortex for 30 seconds, and place them flat at room temperature for 10 minutes; The extraction bufferA described above consists of the following components:

[0036]

[0037] (2) Add 300 μL of buffer B (stored at 4 degrees Celsius), fully invert and mix for 30 seconds, centrifuge at 12,000 rpm for 5 minutes, and transfer the supernatant to another centrifuge tube; the extraction buffer B is KAC (potassium acetate) with a concentration of 5M;

[0038] (3) Add isopropanol 1 times the volume of the supernatant to the supernatant, mix well, centrifuge at 12000rpm for 5min, discard the supernatant, and keep the precipitate;

[0039] (4) The precipitate was dissolved in 1000 μL water (RNase-free) contain...

Embodiment 2

[0044] Example 2: Banana stem total RNA extraction for RNA reverse transcription

[0045] (1) Select 0.5g of fresh and well-grown banana stems, put them into a mortar pre-cooled by liquid nitrogen, add liquid nitrogen to grind them fully until they are powdery, add 500ul to extract buffer A immediately, and vortex for 20 seconds. put for 5min;

[0046] The extraction bufferA consists of the following components:

[0047]

[0048] (2) Add 150 μL of buffer B (stored at 4 degrees Celsius), fully invert and mix for 20 seconds, centrifuge at 12,000 rpm for 10 minutes, and transfer the supernatant to another centrifuge tube; the extraction buffer B is KAC (potassium acetate) with a concentration of 5M;

[0049] (3) Add isopropanol 0.7 times the volume of the supernatant to the supernatant, mix well, centrifuge at 12000rpm for 5min, discard the supernatant, and keep the precipitate;

[0050] (4) The precipitate was dissolved in 500 μL water (RNase-free) containing 20ug / ml DNaseI...

Embodiment 3

[0054] Embodiment 3: Peanut pod total RNA extraction is used for RT-PCR experiment

[0055] (1) Select fresh and tender peanut pods in good growth condition, put them into a mortar pre-cooled by liquid nitrogen, add liquid nitrogen and grind them thoroughly until they are powdery, immediately add 750ul to extract buffer A, vortex for 25 seconds, and place them flat at room temperature 7.5min;

[0056] The extraction bufferA consists of the following components:

[0057]

[0058]

[0059] (2) Add 150 μL of buffer B (stored at 4 degrees Celsius), fully invert and mix for 25 seconds, centrifuge at 12,000 rpm for 7.5 minutes, and transfer the supernatant to another centrifuge tube; the extraction buffer B is KAC (potassium acetate) with a concentration of 5M

[0060] (3) Add isopropanol 0.8 times the volume of the supernatant to the supernatant, mix well, centrifuge at 12000rpm for 5min, discard the supernatant, and keep the precipitate;

[0061] (4) The precipitate was di...

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Abstract

The invention belongs to the technical field of biology and discloses an extraction method of plant RNA (ribonucleic acid). The method comprises the following steps: sufficiently grinding the plant material in liquid nitrogen to powder, adding an extraction buffer A, sufficiently reacting, adding an extraction buffer B, evenly mixing to react, precipitating with isopropanol, separating out the RNA, washing with ethanol, and dissolving in RNase-free water. The method does not need to use DEPC or the expensive extraction kit, and does not need to use phenol, chloroform and other toxic irritating reagents; and the method is simple to operate and easy to implement, has short extraction time (not more than 30 minutes for the whole process) and has the advantages of favorable completeness and higher concentration as compared with the extracted RNA and kit. The method is more suitable for extracting RNA from polyphenol plants from which TRIZOL can not be extracted generally. The purity and concentration of the extracted RNA are sufficient for RNA reverse transcription, RT-PCR (reverse transcription-polymerase chain reaction) and Nothern blot hybrid experiments.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for extracting RNA from plant materials rich in polysaccharides and polyphenols, in particular to a method for quickly and easily extracting RNA from plants rich in polysaccharides and polyphenols using a new extract formula A new method for extracting RNA from leaves, roots, stems, flowers, fruits, and seeds. Background technique [0002] The extraction of RNA from plant tissues is a necessary prerequisite for the study of plant molecular biology. For Northern hybridization analysis, purification of mRNA for in vitro translation or establishment of cDNA library, molecular biology research such as RT-PCR and differential analysis, all require high-quality RNA. Therefore, extracting RNA with high purity and good integrity from plant tissues is the key to the smooth conduct of the above research. [0003] According to literature reports, there are many plants rich in polysac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘海燕梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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