Broad-spectrum high-efficiency plant RNA extracting kit

A kit and plant technology, applied in the field of biochemistry, can solve the problems of insufficient RNA quality and unsatisfactory extraction, and achieve the effects of high yield, low cost and simple operation steps.

Inactive Publication Date: 2011-02-02
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both of these methods have disadvantages in practical applications: first, the extraction success rate can only reach 60%, and second, the quality of extracted RNA is not high enough to meet the requirements of some analysis researches that require high RNA quality, such as RNA expression profiling and sequencing. need

Method used

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  • Broad-spectrum high-efficiency plant RNA extracting kit
  • Broad-spectrum high-efficiency plant RNA extracting kit
  • Broad-spectrum high-efficiency plant RNA extracting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: The inventive method extracts cotton ovule total RNA

[0053] 1. Add 3.0 mL of extraction buffer solution I, 0.25 mL of β-mercaptoethanol, 2.0 mL of Tris saturated phenol with a pH of 8.0, and 1.5 mL of chloroform: isoamyl alcohol with a volume ratio of 24:1 in a 10 mL centrifuge tube;

[0054] 2. Take 0.1g of cotton ovule, add 0.1g of cross-linked polyvinylpyrrolidone PVPP, fully grind into powder in liquid nitrogen, quickly transfer the frozen powder into the above-mentioned 10mL centrifuge tube, vortex vigorously for 3 minutes, and place on ice for 5 minutes;

[0055] Centrifuge at 10,000g for 15min at 3.4°C;

[0056] 4. Transfer the supernatant to a centrifuge tube, add an equal volume of pre-cooled isopropanol, turn over slowly 30 times, mix well, let stand at -20°C for 1-2h, centrifuge at 10,000g for 20min at 4°C, discard the supernatant;

[0057] 5. Add 3mL of extraction buffer solution II to the precipitation, bathe in water at 65°C for 15min; i...

Embodiment 2

[0075] Embodiment 2: Extraction of banana pulp, pericarp, masson pine, cedar RNA. The steps of the experimental method are exactly the same as in Example 1. see results Figure 7 .

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Abstract

The invention discloses a broad-spectrum and high-efficiency method for extracting RNA from various plant tissues and belongs to the technical field of biochemistry. In the invention, boric acid buffer solution is used as a buffer system, the plant tissue RNA is effectively separated and enriched first, the mixture of tris(hydroxymethyl)aminomethane (tris), hydrochloric acid (HCl), ethylenediamine tetraacetic acid disodium salt (EDTA-Na2) and sodium chloride (NaCl) is used as extraction buffer solution, hexadecyl trimethyl ammonium bromide (CTAB) is used as a detergent, 1 mol / L guanidinium isothiocyanate is used as a strong denaturant for inhibiting the activity of RNase, and thus high-quality RNA is extracted. The method can extract RNA from tissues such as ovules, anthers, blades and fibers of cotton and can extract high-quality RNA from recalcitrant plants such as arabidopsis thaliana, rice, draba matangensis, banana (peel and meat), masson pine and deodar. When the method is used for extracting RNA, the yield is high, the purity is high, and salt pollutant is avoided.

Description

1. Technical field [0001] The invention relates to a broad-spectrum and high-efficiency method for extracting plant RNA, which belongs to the field of biochemical technology and is suitable for extracting high-quality RNA from various plants, including recalcitrant plant tissues rich in polysaccharides, polyphenols and secondary metabolites. total RNA. 2. Background technology [0002] The extraction of RNA from plant tissues is a necessary prerequisite for the study of plant molecular biology. For Northern hybridization analysis, purification of mRNA for in vitro translation or construction of cDNA library, large-scale transcriptome and miRNA sequencing analysis, gene chip analysis, RT-PCR and differential analysis and other functional genomics and molecular biology research, all need High quality intact RNA. Therefore, extracting RNA with high purity and good integrity from plant tissues is the basic premise and key step for the above-mentioned research to be carried out...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘康李茂峰王晋惠颖
Owner NANJING AGRICULTURAL UNIVERSITY
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