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Process of extracting total RNA from sycamore tissue

A technology of sycamore and tissue, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of increased difficulty in RNA extraction, different difficulty in extracting RNA, high activity, etc., and achieves improved preparation efficiency and low price , high quality effect

Inactive Publication Date: 2007-06-27
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are some mature total RNA isolation methods that can successfully extract RNA from different plants or different tissues, the difficulty of extracting RNA from different materials or different parts of the same material is different, so it is necessary to focus on the specific characteristics of biological materials. Choosing the Appropriate RNA Extraction Method
In Platanus plant tissue, not only the content of phenolic compounds and secondary metabolites is higher, but also the activity of RNase is higher, these factors make RNA extraction more difficult

Method used

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  • Process of extracting total RNA from sycamore tissue
  • Process of extracting total RNA from sycamore tissue
  • Process of extracting total RNA from sycamore tissue

Examples

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Effect test

Embodiment 1

[0045] Embodiment 1: extract total RNA sample from Platanus inflorescence

[0046] a. Crude extraction of total RNA from Platanus inflorescences:

[0047] (1) Sampling: collect fresh Platanus inflorescences, place it in liquid nitrogen, and obtain sample 1;

[0048] (2) Add 3ml of lysate containing 700μl / 100ml β-ME to the centrifuge tube, and preheat in a water bath at 65°C;

[0049] (3) Take 2g of sample 1 in step (1) and add liquid nitrogen to grind it into powder and add it to the centrifuge tube in step (2), shake it for 1-2 minutes to mix it well, put it in a water bath at 65°C for 5 minutes, and cool it down to room temperature naturally to obtain a sample mixture Liquid 2;

[0050] (4) Add an equal volume of chloroform / isoamyl alcohol (volume ratio of chloroform: isoamyl alcohol = 24:1) to the sample mixture 2 obtained in step (3), turn it upside down to make a single phase, and store at 4°C Centrifuge at 12000rpm / min for 10min under conditions, and recover the super...

Embodiment 2

[0061] Embodiment 2: extract total RNA sample from Platanus leaves

[0062] a. Crude extraction of total RNA from Platanus leaves:

[0063] (1) Sampling: collect fresh leaves of Platanus annuus and place them in liquid nitrogen to obtain sample 1;

[0064] (2) Add 3ml of lysate containing 700μl / 100ml β-ME to the centrifuge tube, and preheat in a water bath at 65°C;

[0065] (3) Take 1g of sample 1 in step (1) and add liquid nitrogen to grind it into powder and add it to the centrifuge tube in step (2), shake it for 1-2 minutes to mix it well, put it in a water bath at 65°C for 2 minutes, and cool it down to room temperature naturally to obtain a sample mixture Liquid 2;

[0066] (4) Add an equal volume of chloroform / isoamyl alcohol (volume ratio of chloroform: isoamyl alcohol = 24:1) to the sample mixture 2 obtained in step (3), turn it upside down to make a single phase, and store at 4°C Centrifuge at 12000rpm / min for 10min under conditions, and recover the supernatant;

...

Embodiment 3

[0077] Example 3: Extraction of total RNA from mixed inflorescences of Platanus azukiensis sampled and preserved in different periods

[0078] a. Rough mention:

[0079] (1) Sampling: in different periods (the samples were taken from February, July, August of the current year and October and December of the previous year), the inflorescences of Platanus chinensis were collected and placed in liquid nitrogen, and stored in a -70°C ultra-low temperature refrigerator for later use. Mixed samples from different periods 1;

[0080] (2) Add 3ml of lysate solution containing 700μl / 100ml β-ME to the centrifuge tube, and preheat in a water bath at 65°C;

[0081] (3) Take 2g of the mixed sample 1 in step (1), add liquid nitrogen and grind it into powder, add it to the centrifuge tube in step (1), shake it for 1-2 minutes to mix it well, put it in a water bath at 65°C for 2 minutes, and cool it naturally to room temperature to obtain the sample Mixture 2;

[0082] (4) Add an equal vol...

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Abstract

The present invention relates to molecular biology technology, and is especially process of extracting total RNA from sycamore tissue. Aiming at overcoming the influence of phenols and secondary metabolites in sycamore tissue on the extraction of high quality RNA sample, the present invention proposes the key steps from initial extraction to the purification of total RNA in sycamore, prepares new cracking solution for inhibiting the oxidation of polyphenol in sycamore tissue, extracts with chloroform / isoamyl alcohol and chloroform separately to eliminate partial polysaccharide, polyphenol and protein, purifies the initial extract dissolved in DEPC-H2O with n-butyl alcohol and water saturated CTAB, so as to raise the efficiency of extracting total RNA from sycamore tissue. The present invention provides new method for the further molecular biology research of sycamore plant.

Description

technical field [0001] The invention relates to the technical field of plane tree molecular biology. It specifically relates to a method for extracting and purifying RNA used in molecular biology from sycamore tissue. Background technique [0002] RNA extraction is the basis for molecular biology research. The acquisition of high-quality RNA is of great significance for functional genomics and genetics research. The extraction method of relevant plant RNA is more, and existing method mainly contains Trizol Kit method (Trizol reagent.Product descriptionTRIZOL reagent.Manufacturer protocol (1995) Life Technologies, Gaithersberg, MD.); Isothiocyanate guanidine method (Chomczynski P and Sacchi N(1987)Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction.Anal Biochem 162:156-159.); SDS / phenol method (Gehrig HH, Winter K, Cushman J, Borland A, and Taybi T (2000) An improved RNA isolation method for succulent plant species richin polyphe...

Claims

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Application Information

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IPC IPC(8): C12N15/29
Inventor 包满珠李志能刘国锋张俊卫黄文俊
Owner HUAZHONG AGRI UNIV
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