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Reagent composition for separating total RNA in plant or microorganism and preparation method thereof

A composition and plant technology, applied in the field of molecular biology, can solve the problems of complicated operation, strong sample selectivity, low efficiency, etc., and achieve the effects of flexible operation, shortening operation time, and overcoming strong selectivity.

Inactive Publication Date: 2012-05-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcomings of traditional methods such as strong sample selectivity, cumbersome operation, and low efficiency, and provide a reagent composition for isolating total RNA of citrus plants, tobacco leaves or pear rot pathogens and a preparation method thereof

Method used

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  • Reagent composition for separating total RNA in plant or microorganism and preparation method thereof
  • Reagent composition for separating total RNA in plant or microorganism and preparation method thereof
  • Reagent composition for separating total RNA in plant or microorganism and preparation method thereof

Examples

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Effect test

Embodiment 1

[0045] Embodiment 1 Application example of the present invention Application of RT-PCR to amplify Mexican strain lemon (Citrus aurantifolia) Actin gene fragment

[0046] In this example, the leaves of the Mexican lemon were collected from the National Fruit Tree Germplasm Resources Indoor Preservation Center of Huazhong Agricultural University, and the Mexican lemon plants were potted plants in the greenhouse. This variety is a commercial variety and a variety (material) widely used in production. If the public needs it, the National Fruit Tree Germplasm Resources Indoor Preservation Center of Huazhong Agricultural University can distribute the material of this variety to the outside world.

[0047] 1. Preparation of reagent composition for RNA extraction

[0048] 1) Glycogen solution (10mg / ml)

[0049] The glycogen powder was dissolved in deionized water treated with DEPC (diethylpyrocarbonate), and the volume was adjusted to 15 mg / ml. Use DNase (purchased from Treasure Dal...

Embodiment 2

[0141] Example 2 Using the reagent composition of the present invention to rapidly extract total RNA from leaves of different citrus varieties

[0142] In this embodiment, the following citrus plants such as sour orange (Citrus aurantium), sweet orange (Citrus sinensis), grapefruit (Citrus paradisi), citron (Citrus medica), Wenzhou tangerine (Citrus unshiu), citrus aurantium (Poncirus trifoliate) and other citrus samples were collected from the National Fruit Tree Germplasm Resources Indoor Preservation Center of Huazhong Agricultural University. These varieties are very common production varieties (or materials) or commercial varieties (or materials). The indoor preservation center of virus-free germplasm resources can distribute the above-mentioned varieties or materials to the outside world.

[0143] 1. Preparation of RNA reagent composition

[0144] 1) Glycogen solution (10mg / ml)

[0145] After the glycogen powder was dissolved in deionized water treated with diethyl pyr...

Embodiment 3

[0180] Example 3 Rapid Extraction of Ben's tobacco (Nicotiana benthamiana) leaf total RNA using the reagent composition of the present invention

[0181] In this example, the leaves of N. benthamiana were collected from the National Center for Virus Removal and Germplasm Resources Indoor Preservation Center of Huazhong Agricultural University.

[0182] 1. Solution preparation

[0183] The following reagent compositions need to be prepared for RNA extraction:

[0184] 1) Glycogen solution (10mg / ml)

[0185] After the glycogen powder was dissolved in deionized water treated with diethyl pyrocarbonate (DEPC), the volume was adjusted to 15 mg / ml. DNAse (purchased from TaKaRa Company), RNase (purchased from TaKaRa Company) and proteinase K (purchased from TaKaRa Company) were used for digestion successively to remove DNA, RNA and protein contamination in the medicine. Use an equal volume of water-saturated phenol to extract once, and after extracting twice with an equal volume o...

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Abstract

The invention belongs to the field of molecular biology method and relates to a reagent composition for separating total RNA in a plant or a microorganism and a preparation method thereof. The high quality RNA can be obtained after a simple extraction by chloroform while guanidinium isothiocyanate and phenol are regarded as main components, positive ions are provided by NaCl, MgCl2 and the like, and a solution pH is stabilized by a sodium acetate-acetic acid buffer system. Glycogen serving as a nucleic acid precipitant is added in the RNA extraction reagent in the invention, so that the reagent disclosed by the invention, in comparison with reagents of the same type, greatly improves precipitation efficiency of the nucleic acid and also can efficiently separate the nucleic acid component which has a very low content in a tissue sample. Therefore, the nucleic acid precipitation process can be finished in a short period, the precipitation process at -20 DEG C for hours is avoided, and operating time is shortened greatly. The method is rapid as well as efficient and has wide applicable samples; and the reagent composition disclosed by the invention is cheaper than commercial TRIzol reagents. The disadvantages of high sample selectivity, fussy operation and relatively low efficiency of the traditional method are overcome. The operation is more flexible; and the sample after being homogenized can be stored at -20 DEG C and then used for the RNA extraction.

Description

technical field [0001] The invention relates to the field of molecular biology, and relates to a method for preparing total RNA of cells, in particular to a reagent composition for isolating total RNA of citrus plants, tobacco leaves or pear rot pathogen and a preparation method thereof. Background technique [0002] RNA is the main component involved in gene expression in cells, and plays a very important role in the maintenance and expression of genetic information. In modern molecular biology research, RNA isolation has become an indispensable technical means for many experiments. The quality of the isolated RNA will also directly affect the follow-up experiments. Due to its own biological characteristics, RNA is easy to degrade and difficult to preserve. The extraction process is also prone to contamination by proteins, DNA, and other cellular metabolites. Therefore, a method that can isolate complete and high-purity RNA has always been the goal that molecular biology...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 杨帆洪霓王国平王利平徐文兴丁芳
Owner HUAZHONG AGRI UNIV
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