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Kit for extracting RNA and application method

A kit and final concentration technology, applied in the field of molecular biology, can solve problems such as cumbersome centrifugation steps, time-consuming, physical and environmental hazards for operators, and achieve improved throughput and efficiency, high integrity, and labor-saving Effect

Active Publication Date: 2018-12-07
广州奇辉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Advantages of TRIzol method: low extraction cost, the most classic and common RNA extraction method. Disadvantages: cumbersome operation steps, long time-consuming, low purity of RNA extraction, and most of the reagents used are toxic, which may cause harm to the operator's body and the environment
[0006] The more common column-passing method in the reagent method is combined with the TRIzol method, adding chloroform for layering, and then extracting the aqueous phase RNA through the column. Compared with the TRIzol method, the purity of RNA extraction is higher, but the steps such as centrifugation are too cumbersome, and the reagents are also harmful to operators and the environment.

Method used

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  • Kit for extracting RNA and application method
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  • Kit for extracting RNA and application method

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1 RNA extraction kit of the present invention extracts the method for whole blood RNA

[0066] Take 5 parts of fresh whole blood, each 200 μl, use the RNA extraction kit of the present invention to extract the specific operation as follows:

[0067] (1) Add all 200 μl samples into a 1.5ml nuclease-free centrifuge tube;

[0068] (2) Add 200 μl of lysate and 10 μl of proteinase K (10 mg / ml) to the centrifuge tube of (1), shake and mix;

[0069] (3) Add 300 μl of isopropanol and 15 μl of extraction magnetic beads to (2), and mix at room temperature for 10 minutes;

[0070] (4) Centrifuge the centrifuge tube in (3) for about 5 seconds, place it on the magnetic stand for 3 minutes, and discard the liquid;

[0071] (5) Add 400 μl of washing solution I to the centrifuge tube in (4), vortex and mix, centrifuge briefly, place on the magnetic stand for 2 minutes, and discard the liquid;

[0072] (6) Prepare DNase I and DNase I Buffer mixture according to the followi...

Embodiment 2

[0080] Example 2 TRIzol method and the comparison effect verification of RNA kit of the present invention to extract tissue RNA

[0081] A comparative experiment was designed to compare the traditional TRIzol method on the market with the RNA kit of the present invention to extract tissue RNA yield, purity, integrity, hazards to personnel and the environment, etc.

[0082] The mouse liver was taken, cut, weighed, and equally divided into 10 parts, 30 mg each; randomly divided into two groups, 5 parts in each group. 5 RNAs were extracted respectively according to the magnetic bead method extraction kit of the present invention and the traditional TRIzol method.

[0083] The extraction operation method of the RNA kit extracted by the magnetic bead method of the present invention is as follows:

[0084] (1) Add the sample to a 1.5ml nuclease-free centrifuge tube,

[0085] (2) Add 200 μl of lysate to (1) centrifuge tube, pipette to evacuate the tissue, add 400 μl of tissue diges...

Embodiment 3

[0114] Example 3 Effect verification of lithium chloride and glycogen concentration in RNA extraction

[0115] Experiments were designed to verify the effect of different concentrations of lithium chloride and glycogen in the lysate on the RNA extraction effect. Prepare the lysate according to the lysate preparation component requirements and the concentration range of the present invention: 3mol guanidine isothiocyanate, 10mmol / L sodium citrate, 2% Tween-20 by volume, 1% potassium acetate by volume, final The concentration is 10mM DTT; the glycogen concentration is set to 0mg / mL, 0.1mg / mL, 0.2mg / mL, 0.5mg / mL, 1mg / mL five concentration gradients; the lithium chloride concentration is set to 0mol / L, 0.5 mol / L, 1mol / L, 2mol / L four concentration gradients. After the respective concentrations of the two components were combined, a total of 20 concentration combinations were produced.

[0116] According to the method described in Example 1, RNA was extracted from the whole blood ...

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Abstract

The invention belongs to the field of molecular biology and particularly relates to a kit for extracting ribonucleic acid (RNA) by virtue of a paramagnetic particle method and an extraction method. The kit contains tissue digestion fluid, lysate, proteinase K, DNase I, DNase IBuffer, nucleic acid, extraction magnetic beads, washing liquid I, washing liquid II and eluant. The invention further discloses a method for extracting the tissue RNA by virtue of the kit. According to the kit and the method, the extraction yield and purity of RNA are increased, the integrity of RNA is improved, meanwhile, the automatic extraction is realized, and the simultaneous parallel testing of multiple samples is realized, so that the labor cost and the time cost are saved.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a kit and an extraction method for extracting ribonucleic acid (RNA) in biological tissue samples. Background technique [0002] RNA is a single strand formed by transcription based on a strand of DNA as a template and based on the principle of complementary base pairing. [0003] RNA extraction is easy to degrade, and RNA is easy to degrade due to internal and external factors. From its own structure, RNA is a single-stranded molecule, which is very unstable. In addition, there is a free hydroxyl group at the 2' position of the RNA molecule, which is easy to form 2' , 3'-cyclic phosphate intermediate, easy to decompose. From the perspective of external factors, RNase is ubiquitous, and RNase itself has high thermal stability and is not easy to be inactivated. Therefore, during the RNA extraction process, all utensils that come into contact with RNA must be RNase-free, such as D...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2521/537
Inventor 朱奇王灵敏廖传荣刘丽
Owner 广州奇辉生物科技有限公司
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