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Method for extracting RNA of sweet potato root tuber and application thereof

A technology of root tubers and sweet potatoes, applied in the field of molecular biology, can solve the problems of high toxicity of lysate, time-consuming and laborious, unsatisfactory results, etc., and achieve the effect of high purity, large quantity and good integrity

Inactive Publication Date: 2010-07-21
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to avoid starch swelling and gelatinization, Mohan Kumar et al. (2007) extracted total RNA from sweet potato tubers by SDS and high-salt precipitation methods. However, these methods may have the disadvantages of high toxicity of the lysate used or time-consuming and labor-intensive problems.
In recent years, some total RNA extraction reagents (boxes) have been developed successively at home and abroad for the RNA extraction of special plant materials rich in polysaccharides and polyphenols, but the results are not satisfactory.

Method used

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  • Method for extracting RNA of sweet potato root tuber and application thereof
  • Method for extracting RNA of sweet potato root tuber and application thereof
  • Method for extracting RNA of sweet potato root tuber and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Preparation of reagents

[0041] A. Preparation of DEPC water: Add 0.1% (volume ratio) DEPC to double-distilled water, stir for more than 4 hours, place at 37°C for overnight treatment, and sterilize at 121°C for 20 minutes;

[0042] B. 1mol / L Tris-HCl (pH9.0): add 12.114g Tris to 100ml sterilized DEPC water, adjust the pH value to 9.0 with concentrated hydrochloric acid;

[0043] C, 0.5mol / L EDTA (pH8.0): 18.61g EDTANA 2 .H 2 O, dissolve in unsterilized DEPC water, make up to 100ml, adjust the pH value to 8.0 with NaOH particles, and sterilize at 121°C for 20min;

[0044] D. 8mol / L liCl: Dissolve 33.912g LiCl in unsterilized DEPC water, dilute to 100ml, and sterilize at 121°C for 20min;

[0045] E. 4mol / L NaCl: Dissolve 23.26g NaCl in unsterilized DEPC water, dilute to 100ml, and sterilize at 121°C for 20min;

[0046] F. 5mol / L potassium acetate: Dissolve 49.07g potassium acetate in unsterilized DEPC water, dilute to 100ml, and sterilize at 121°C for 20min;

...

Embodiment 2

[0069] (1) Preparation of reagents

[0070] A. RNA extraction buffer (prepared 100ml): 5ml 1mol / L Tris-HCl (pH9.0), 25ml 4mol / L NaCl, 5ml 0.5mol / L EDTA (pH8.0), 1g CTAB and 1g PVP and mix well, Then the volume was adjusted to 66ml with sterilized DEPC water, and 33ml of water-saturated phenol (pH4.3) and 1ml of β-mercaptoethanol were added before use.

[0071] B. Polysaccharide removal solution (100ml prepared): prepared by mixing 10ml absolute ethanol, 8ml 5M potassium acetate and 82ml sterilized DEPC water.

[0072] (2) Extraction of total RNA in the later stage of sweet potato root development

[0073] The same as the operation of step (2) in Example 1, the only difference is that the sample used is 1 g of sweet potato tuber tissue in the later stage of development (140 days of growth period). The extracted total RNA was tested by 1.0% agarose gel electrophoresis, and the 28S, 18S, and 5.8S band patterns were clear, indicating that the integrity of the total RNA obtained ...

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Abstract

The invention discloses a method for extracting RNA of sweet potato root tuber and application thereof. Beta-mercaptoethanol and polyvinylpyrrolidone are used to prevent polyphenols from interfering the separation of RNA; and in addition, the NaCl with high concentration, absolute ethyl alcohol and potassium acetate are used, so the polysaccharose can be rapidly precipitated, and the influence of the polysaccharose on the extraction of RNA can be prevented. When preventing the interference effect of the polyphenols and the polysaccharose on the separation of RNA, RANA can still be remained in the solution, so the RNA with good quality, high purity and good completeness can be obtained, and the attainment rate and the yield of the RNA are large. The method has high practical application value, and can satisfy the research of molecular biology such as reverse transcription, Northern hybridization, cDNA library construction, transcriptome analysis and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for extracting RNA from sweet potato tubers and its application. Background technique [0002] Sweet potato is an important industrial raw material, biomass energy and health food. Therefore, people urgently need to understand the genetic information of sweet potato more deeply, and extracting high-purity and high-quality RNA is the necessary prerequisite and key to understand the genetic information of sweet potato. [0003] Sweet potato roots are rich in polysaccharides (mainly starch), phenols and other secondary metabolites, which seriously affect the extraction of RNA. Among them, the physical and chemical properties of polysaccharides are very similar to those of ribonucleic acid RNA. When precipitating RNA, proteoglycan and RNA are precipitated together to form an insoluble colloidal complex. When polysaccharides are removed, RNA is also taken away, resulting in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王章英房伯平陈景益张雄坚罗忠霞黄立飞陈新亮温少旋
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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