Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites

A plant tissue and secondary metabolism technology, applied in the field of plant molecular biology, can solve the problems of long time, many processing steps, and the ineffectiveness of conventional methods, and achieve the effect of improving quality and inhibiting obstacles.

Inactive Publication Date: 2010-02-03
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Many plants, especially their fruits, are rich in secondary metabolites such as aromatic phenolic compounds, polysaccharide compounds, and flavonoids, making the extraction of nucleic acids very difficult
Especially for the extraction of total RNA, conventional methods are difficult to work
Although some studies have reported that acetone can remove some flavonoid

Method used

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  • Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites
  • Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites
  • Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Extraction and application of total RNA from young leaves (red), colored pericarp and uncolored pericarp of Yunnan Hongpi pear

[0039] (1) Take 1 g each of the young leaves (red), colored peel, and uncolored peel of Yunnan red-skinned pears quick-frozen in liquid nitrogen, and grind them into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 10mL pre-cooled RNA extraction buffer, mix well and place on ice for 5min, then add 3mol L of pH4.5 -1 Sodium acetate 1mL, mix upside down;

[0040] (2) Then add 11 mL of chloroform, shake vigorously to mix the two phases, place on ice for 10 min, then centrifuge at 12,000 g at 4°C for 10 min, and take the supernatant into a new centrifuge tube;

[0041] (3) Repeat the chloroform extraction step in (2) once;

[0042] (4) Add 10mL of isopropanol to the supernatant, mix well, place at -20°C for 30min, then centrifuge at 12000g at 4°C for 15min to obtain RNA precipitation;

[0043] (5...

Embodiment 2

[0079] Embodiment 2: grape young leaves, young fruit pericarp, pulp total RNA extraction

[0080] (1) Take 1.2 grams each of liquid nitrogen quick-frozen grape leaves, young fruit peels, and pulp, and grind them into powder with liquid nitrogen; quickly transfer each sample to a centrifuge tube, and add 12 mL of pre-cooled RNA extraction buffer , mix well and place on ice for 5min, then add 3mol L of pH4.5 -1 Sodium acetate 1.2mL, mix up and down;

[0081] (2) Then add 13mL chloroform, shake vigorously to mix the two phases, place on ice for 10min, centrifuge at 12000g at 4°C for 10min, take the supernatant into a new centrifuge tube;

[0082] (3) Repeat the chloroform extraction step in (2) once;

[0083] (4) Add 12mL of isopropanol to the supernatant, mix well, place at -20°C for 30min, then centrifuge at 12000g at 4°C for 15min to obtain RNA precipitation;

[0084] (5) Wash the precipitate twice with 75% ethanol, dry at room temperature for 20min, and then use 1.2mL DEPC...

Embodiment 3

[0092] Embodiment 3: strawberry immature fruit (green) and ripe fruit (red) total RNA extraction

[0093] (1) Take liquid nitrogen quick-frozen strawberry immature fruit and 2 grams of mature fruit, and grind it into powder with liquid nitrogen; quickly transfer each sample to a centrifuge tube, and add 20 mL of pre-cooled RNA extraction buffer, Mix well and place on ice for 5min, then add 3mol L of pH4.5 -1 Sodium acetate 2mL, mix up and down;

[0094] (2) Then add 22mL of chloroform, shake vigorously to mix the two phases, place on ice for 10min, then centrifuge at 12000g at 4°C for 10min, and take the supernatant into a new centrifuge tube;

[0095] (3) Repeat the chloroform extraction step in (2) once;

[0096] (4) Add 20mL of isopropanol to the supernatant, mix well, place at -20°C for 30min, then centrifuge at 12000g at 4°C for 15min to obtain RNA precipitation;

[0097] (5) Wash the precipitate twice with 75% ethanol, dry it at room temperature for 20 min, and then u...

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Abstract

The invention provides an extration method of plant RNA by utilizing guanidinium isothiocyanate-chloroform extractive and Tris purification. The invention improves traditional guanidinium isothiocyanate method, adopts the two steps of chloroform extration after cracking and phenol-adding for purification, and respectively extract high-quality total RNA from the leaf blade and seed case of red-skinned pears, the leaf blade, fruit flesh and seed case tissues of grapes, the fruit of strawberries and the callus of arnebia euchroma. The extraction buffer liquid in the method contains guanidinium isothiocyanate, sodium dodecyl sarcosinate, sodium citrate, soluble polyvinylpyrrolidone and beta-mercaptoethanol, which can not only crack plant cells efficiently and release RNA, but also effectivelyinhibit the activity of RNA enzyme, and can also prevent the oxidation of phenolic compounds and suppress the interference of secondary metabolites. In the second step, Tris purification is utilized for crude extraction of RNA solution, which can eliminate the polysaccharides and protein which can coprecipitate with RNA. The invention is simple in operating steps, economic and practical, good in stability, and suitable for extracting high-quality total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites.

Description

technical field [0001] The invention relates to the technical field of plant molecular biology, in particular to a method for extracting and purifying total RNA from plant tissues rich in polysaccharides, polyphenols and secondary metabolites. Background technique [0002] The extraction of RNA is one of the basic contents of molecular biology. High-quality RNA is a necessary prerequisite for gene cloning, gene expression, and gene function analysis. For molecular biology research such as RT-PCR, Northern, real-time PCR, and cDNA library construction, total RNA with high purity and integrity must first be obtained. The complex and diverse components of plant tissues, especially higher plant tissues, make the extraction of RNA from plant tissues more difficult than other biological materials. For various plants of the genus Pyrus, Citrus, and Chinese herbal medicine, it is difficult to isolate and purify RNA in tissues, which seriously hinders the progress of their molecular...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/02
Inventor 刘迪秋王继磊李文娴葛锋陈朝银田荣欢
Owner KUNMING UNIV OF SCI & TECH
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