Method for quickly extracting total ribonucleic acid (RNA) from Rubus

A technology of Rubus genus and an extraction method, applied in the field of plant molecular biology, can solve problems such as long time consumption, DNA pollution, RNA loss, etc., and achieve the effects of short time consumption, increased applicability, and simplified operation process

Inactive Publication Date: 2012-01-25
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest disadvantage of this method is that it takes too long, and generally choose to settle overnight
If it is changed to alcohol precipitation, the DNA pollution is particularly serious, and further treatment with DNase is required, followed by phenol-chloroform extraction to inactivate DNase, resulting in a large loss of RNA
In addition, the elution process after LiCl precipitation is strictly required, because the residual Li + , Cl - Will have a negative impact on subsequent reverse transcription and PCR amplification

Method used

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  • Method for quickly extracting total ribonucleic acid (RNA) from Rubus
  • Method for quickly extracting total ribonucleic acid (RNA) from Rubus
  • Method for quickly extracting total ribonucleic acid (RNA) from Rubus

Examples

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Effect test

Embodiment 1

[0038]1) Take a 2ml centrifuge tube, add 1000 μl of sugar-free buffer to the centrifuge tube, add 5 μl of β-mercaptoethanol, and pre-cool at -20°C for 10 minutes; 0.15g, add 0.1g cross-linked polyvinylpyrrolidone, grind into powder under liquid nitrogen freezing conditions, quickly transfer to the centrifuge tube in step 1), mix gently up and down, and let stand on ice for 10min; 3) Centrifuge at 3200×g for 10 min at 4°C; 4) Completely remove the supernatant obtained in step 3), add 700 μl of cell lysate preheated at 65°C to the pellet, vortex for 1 min, and incubate at 60°C for 30 min; 5) After the mixture in step 4) is cooled to room temperature, add 700 μl of water-saturated phenol, 13 μl of glacial acetic acid, and 140 μl of chloroform in sequence, and shake vigorously for 2 minutes; 6) centrifuge at 16,000×g for 10 minutes at 4°C; ) and transfer 500 μl of the supernatant obtained by centrifugation into another centrifuge tube, add 500 μl of -20°C pre-cooled isopropanol to...

Embodiment 2

[0041] 1) Take a 2ml centrifuge tube, add 700μl lysis buffer to the centrifuge tube, add 5μl β-mercaptoethanol, and preheat at 60°C for 10min; 2) Take 0.1-0.15g of fresh raspberry leaves, add 0.1g Vinylpyrrolidone was ground into powder under liquid nitrogen freezing conditions, quickly transferred to the centrifuge tube in step 1), vortexed for 1 min, and kept at 60°C for 30 min; 3) After the mixture in step 2) was cooled to room temperature , add 700 μl of water-saturated phenol, 12 μl of glacial acetic acid, and 140 μl of chloroform in sequence, and shake vigorously for 2 minutes; 4) Centrifuge at 16,000×g for 10 minutes at 4°C; 5) Transfer 500 μl of the supernatant from step 4) to another centrifuge tube, add 1250 μl -20°C pre-cooled absolute ethanol to the tube, mix upside down and stand at -20°C for 30 minutes, centrifuge at 16000×g for 10 minutes at 4°C, discard the supernatant, and keep the precipitate; 6 ) washing the precipitate obtained by centrifugation in step 5) ...

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Abstract

The invention discloses a method for quickly extracting total ribonucleic acid (RNA) from Rubus. The method is characterized by comprising the following steps of: grinding a small amount of Rubus material into powder under liquid nitrogen freezing conditions, and optionally removing sugar and centrifuging, or directly adding an alkaline cell lysis solution and lysing in warm bath; performing phenol-weak acid-chloroform extraction after the lysis is finished, and centrifuging to remove proteins and most of genomic deoxyribonucleic acid (DNA); adding alcohol into supernatant and precipitating; and centrifuging and collecting a RNA precipitate, washing by using ethanol, air-drying, dissolving by using diethylprocarbonate (DEPC) sterile water, and preserving at the temperature of -80 DEG C. The use of a LiCl precipitation method is avoided, the proteins and the genomic DNA are removed through one-step extraction, the RNA is precipitated by using the alcohol, the method is easy to operate,and the whole process is finished within 3 hours; and only 0.1 to 0.2g of material is required when the total RNA is extracted from the Rubus by the method, the optional step of removing the sugar isadded, and the method is applicable to various plant materials of the Rubus such as raspberries, blackberries and the like.

Description

(1) Technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a method for rapidly extracting total RNA of plants of the genus Rubus (2) Background technology [0002] Obtaining high-quality total RNA is a prerequisite for molecular biology research such as gene expression level research, genetic manipulation such as RNA interference, transgenic and other downstream research. Raspberries and blackberries of Rubus plants are rich in polyphenolic secondary metabolites such as anthocyanins and proanthocyanidins. These substances bring many difficulties to the extraction of total RNA: phenolic compounds are easily oxidized into The brown material then binds irreversibly to RNA, leading to loss of RNA activity, or forms insoluble complexes leading to substantial loss of RNA upon extraction with chloroform. [0003] Commercial kits, such as Trizol kits, are powerless when dealing with the extraction of total RNA from such p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 汤浩茹陈清刘泽静王小蓉余昊唯贾慧峰
Owner SICHUAN AGRI UNIV
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