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Method for extracting RNA in gymnosperm tissue

A gymnosperm and tissue technology, applied in the field of extracting RNA from arboreal plant tissues, can solve the problem of gymnosperm molecular biology and genetic engineering forest tree resistance, reproductive development, material improvement lagging behind, lagging behind, and inability to extract high-quality RNA and other issues to achieve the effect of great practical application value

Inactive Publication Date: 2009-07-29
BEIJING FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the genetic background of gymnosperms and the practical difficulties in molecular manipulation, the molecular biology and genetic engineering of gymnosperms, including tree resistance, reproductive development, and material improvement, are obviously lagging behind.
At present, there are many methods for RNA extraction, and methods such as Trizol kit, SDS, and conventional CTAB cannot effectively extract high-quality RNA from woody gymnosperm tissues
So far, there is no effective method and technology for extracting high-quality RNA from various tissues of woody gymnosperms, which leads to a lag in the development and application of molecular biology research and biotechnology related to gymnosperms compared to angiosperms and annual crops

Method used

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  • Method for extracting RNA in gymnosperm tissue
  • Method for extracting RNA in gymnosperm tissue
  • Method for extracting RNA in gymnosperm tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, extracting the total RNA (not removing DNA) of the needle leaf tissue of P.

[0042] Extracting the total RNA (not removing DNA) of the needle-leaf tissue of P. chinensis comprises the following steps:

[0043] 1. Add 2% (volume percent) β-mercaptoethanol and 10mmol / L dithiothreitol (DTT) to the extraction buffer before use, and place it in a 65°C water bath for 15 minutes. The amount of the sample and the extract is 1:5 (g / mL);

[0044] 2. Grind 2 to 3 g of the needle leaves of Pygnus japonicus in liquid nitrogen, add it to an 80 mL centrifuge tube, vortex rapidly for 30 to 60 seconds, heat at 65°C for 15 minutes, add 15 mL of chloroform / isoamyl alcohol, and mix well;

[0045]3. Centrifuge at room temperature for 15 minutes at 10,000 rpm, transfer the supernatant to another centrifuge tube, add an equal volume of chloroform / isoamyl alcohol and extract again;

[0046] 4. Transfer the upper aqueous phase to another centrifuge tube, add an equal volume of ...

Embodiment 2

[0053] Embodiment 2, extracting total RNA (removing DNA)

[0054] Extracting the total RNA (removing DNA) of the needle-leaf tissue of P. chinensis comprises the following steps:

[0055] In embodiment 2, after step 10) of embodiment 1, a step of removing DNA is added, and the other steps are identical. The steps to remove DNA include the following:

[0056] a) Remove genomic DNA in RNA with DNase without RNase contamination (50 μL reaction system contains 5 μL of 10×DNaseI buffer, 10U DNase, 20U RNase Inhibitor, 20-50 μg RNA) and react at 37°C for 30 minutes;

[0057] b) Extraction with an equal volume of chloroform / isoamyl alcohol to remove impurities such as enzymes in the RNA, and centrifuge at 12000rpm for 30 minutes;

[0058] c) Transfer the upper aqueous phase to a new 1.5ml centrifuge tube, use 2 times the volume of absolute ethanol and 1 / 10 of the volume of 5mol / L potassium acetate, the final concentration of absolute ethanol is 70% (volume percentage), the final co...

Embodiment 3

[0065] Embodiment 3, extracting the total RNA of the white bark pine pollen tissue (not removing DNA)

[0066] The specific steps of extracting the total RNA (without removing DNA) from the white bark pine pollen tissue are as follows:

[0067] 1. Add 2% (volume percent) β-mercaptoethanol 10mmol / L dithiothreitol (DTT) to the extraction buffer before use, and place it in a 65°C water bath for 15 minutes. The amount of sample and extract is 1 : 5 (g / mL);

[0068] 2. Grind 2g of mature pollen of Pinus alba in liquid nitrogen, add it to an 80mL centrifuge tube, vortex rapidly for 30-60 seconds, incubate at 65 for 15 minutes, add 15mL of chloroform / isoamyl alcohol, and mix well;

[0069] 3. Centrifuge at room temperature for 15 minutes at 10,000 rpm, transfer the supernatant to another tube, add an equal volume of chloroform / isoamyl alcohol and extract again;

[0070] 4. Transfer the upper aqueous phase to another centrifuge tube, add an equal volume of 4mol / L LiCl to make the fi...

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Abstract

The invention discloses a method for extracting RNA from gymnosperm tissues. The method includes the following steps: 1) extracting buffer solution by RNA to treat plant materials; 2) protein removal: extracting by equal-volume chloroform / isoamylol for twice; 3) using LiCl to deposit RNA and SSTE to dissolve back RNA; 4) glycan removal: adding absolute ethyl alcohol and potassium acetate to the dissolved solution acquired in step 3) and depositing glycan in ice bath; 5) extracting chloroform / isoamylol again; 6) depositing RNA and DEPC H2O again to dissolve back RNA. In order to remove DNA, the following steps can be also conducted: a) using DNase without RNase pollution to remove DNA; b) using chloroform / isoamylol to extract and remove DNase; (c) repeating step 6). Wherein, RNA extract and buffer solution contains 100mmol / L Tris HCl(pH 8.0), 2g / 100mlCTAB, 2g / 100mlPVP, 2mol / LNaCl and 25mmol / L EDTA and is added with dithiothreitol with final concentration of 10mmol / L and 2 percent (volume percentage) of beta-mercaptoethanol. Therefore, the invention solves the interference of glycan and polyphenol and the like and acquires high-purity RNA.

Description

technical field [0001] The invention relates to a method for extracting RNA from tree plant tissue, in particular to a method for extracting RNA from gymnosperm tissue. Background technique [0002] Gymnosperms and angiosperms belong to seed plants. As an important plant group on the earth, gymnosperms are more primitive in the evolutionary history, with long growth cycles, slow growth of pollen tubes, and easy bifurcation. Therefore, they are considered to be different from angiosperms. The unique developmental model of pollen development has gradually attracted attention. Gymnosperms are closely related to human production and national economic construction, and have high ecological, social and economic benefits. They play a very important role in maintaining the global ecological balance and improving environmental pollution. Most of the gymnosperms are important forest trees, especially in the northern hemisphere, more than 80% of the large forests are gymnosperms, whic...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H1/08C12N15/10
Inventor 张凌云于彦丽郑成超李彦泽
Owner BEIJING FORESTRY UNIVERSITY
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