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Method suitable for rapidly extracting high quality RNA of polysaccharide polyphenol plant tissue

A technology of plant tissue and extraction method, applied in the field of RNA, can solve the problems of high quality of plant tissue RNA, loss of 5SRNA, inappropriate extraction, etc., and achieve the effect of no DNA pollution and high RNA quality.

Inactive Publication Date: 2012-03-28
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Use the plant tissue RNA quality that the present invention extracts to be high, be applicable to the molecular biology experiment relevant to gene expression downstream, but in extraction process, 5S RNA is lost, so the present invention is not suitable for extracting the small RNA (20~20~20) in plant tissue. 25nt)

Method used

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  • Method suitable for rapidly extracting high quality RNA of polysaccharide polyphenol plant tissue
  • Method suitable for rapidly extracting high quality RNA of polysaccharide polyphenol plant tissue
  • Method suitable for rapidly extracting high quality RNA of polysaccharide polyphenol plant tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Application reagent of the present invention and method extract the RNA of banana leaf and root

[0024] (1) Get each 200mg of fresh blade and root from banana seedlings and pulverize in liquid nitrogen;

[0025] (2) Add 1.2mL lysate preheated at 65°C to a 2mL centrifuge tube, add crushed plant tissue with liquid nitrogen, shake and mix;

[0026] (3) Add 600 μL of a mixture of chloroform and isoamyl alcohol, the volume ratio of chloroform and isoamyl alcohol is 24:1, shake and mix, and centrifuge at 8000 r / min at 4°C for 15 min;

[0027] (4) Take the supernatant, add 400 μL of precipitation solution, mix well, and precipitate at -20 ° C for 0.5 to 2 hours, then centrifuge at 12000 r / min below 4 ° C for 15 min;

[0028] (5) Discard the supernatant, precipitate with 500 μL of resuspension solution, add 500 μL of ice-cold water-saturated phenol (pH<5.0), shake and mix well, place on ice for 10 min, below 4°C, 12000 r / min, centrifuge for 15 min;

[0029] (6) ...

Embodiment 2

[0032] Embodiment two: application reagent of the present invention and method extract the RNA of banana pulp

[0033] (1) Get 100 mg of fresh young banana fruit and pulverize it in liquid nitrogen;

[0034] (2) Add 1.2mL lysate preheated at 65°C to a 2mL centrifuge tube, add crushed plant tissue with liquid nitrogen, shake and mix;

[0035] (3) Add 600 μL of a mixture of chloroform and isoamyl alcohol, the volume ratio of chloroform and isoamyl alcohol is 24:1, shake and mix well, centrifuge at 8000 r / min below 4 °C for 15 min;

[0036] (4) Take the supernatant, add 400 μL of precipitation solution, mix well, and precipitate at -20 ° C for 0.5 to 2 hours, then centrifuge at 12000 r / min below 4 ° C for 15 min;

[0037] (5) Discard the supernatant, precipitate with 500 μL of resuspension solution, add 500 μL of ice-cold water-saturated phenol (pH<5.0), shake and mix well, place on ice for 10 min, below 4°C, 12000 r / min, centrifuge for 15 min;

[0038] (6) Take the supernatant...

Embodiment 3

[0041] Embodiment three: application reagent of the present invention and method extract longan pulp RNA

[0042] (1) Get 100 mg of fresh longan pulp and pulverize it in liquid nitrogen;

[0043] (2) Add 1.2mL lysate preheated at 65°C to a 2mL centrifuge tube, add crushed plant tissue with liquid nitrogen, shake and mix;

[0044] (3) Add 600 μL of a mixture of chloroform and isoamyl alcohol, the volume ratio of chloroform and isoamyl alcohol is 24:1, shake and mix well, centrifuge at 8000 r / min below 4 °C for 15 min;

[0045] (4) Take the supernatant, add 400 μL of precipitation solution, mix well, and precipitate at -20 ° C for 0.5 to 2 hours, then centrifuge at 12000 r / min below 4 ° C for 15 min;

[0046] (5) Discard the supernatant, precipitate with 500 μL of resuspension solution, add 500 μL of ice-cold water-saturated phenol (pH<5.0), shake and mix well, place on ice for 10 min, below 4°C, 12000 r / min, centrifuge for 15 min;

[0047] (6) Take the supernatant, add 500 μL...

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Abstract

The invention discloses a method suitable for rapidly extracting high quality RNA of polysaccharide polyphenol plant tissue. The method comprises the following steps: preheating a lysate at the temperature of 65 DEG C, adding plant tissue crushed by liquid nitrogen, oscillating and uniformly mixing, and then adding chloroform and isoamyl alcohol according to ratio of 24:1 for extracting; adding a deposition solution to a supernatant, centrifuging and removing the supernatant; adding a heavy suspension for heavy suspension deposition, respectively extracting by phenol water (pH<5.0) and a mixture of chloroform and isoamyl alcohol according to ratio of 24:1, centrifuging and taking the supernatant; adding anhydrous ethyl alcohol for deposition, washing sediment by 75% of ethanol, air-drying and dissolving by RNase free H2O. The present invention has the advantages that: 1, high quality RNA is successfully extracted from plant tissue containing polysaccharides, polyphenols and secondary metabolites; 2, the operation time is short; 3, no DNA pollution is generated. The main components of the lysate employed in the invention comprise CTAB, Tris-HCl, LiCl, EDTA, NaCl and PVP40; the main component of the deposition solution is LICL; the main component of the heavy suspension is NaCl.

Description

technical field [0001] The invention relates to a reagent and a method for rapidly extracting high-quality RNA from plant tissues, in particular to extracting high-quality RNA from polysaccharide and polyphenol plant tissues including plant fruits. Background technique [0002] It is difficult to extract high-quality RNA from plant tissues rich in polysaccharides, polyphenols and secondary metabolites. At present, cold phenol method, hot phenol method and LiCl precipitation method are generally used. The cold phenol method has many steps, and the whole process needs to be kept at low temperature; while the LiCl precipitation method requires overnight precipitation, which is time-consuming and inefficient. Therefore, finding a method suitable for rapid extraction of high-quality RNA from plant tissues rich in polysaccharides, polyphenols and secondary metabolites can lay a good foundation for the study of such plant molecular biology. Most commercial kits use the TRIZOL meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 彭明阮孟斌李文彬于晓玲
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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