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Method for separating mRNA by using gold magnetism particles

A technology of gold magnetic particles and magnetic particles, which is applied in the field of separating mRNA by using gold magnetic particles, can solve the problems of high price, methods that do not involve mRNA separation, unfavorable general use in general laboratories, etc., and achieves reduction of separation costs and immobilization operations Time saving, high fixed capacity effect

Active Publication Date: 2010-05-12
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Invitrogen, Promega and other foreign companies have listed corresponding mRNA isolation products, but their price is expensive, which is not conducive to the general use of general laboratories
[0005] The preparation method and structural composition of assembly-type magnetic composite particles and core / shell-type superparamagnetic composite particles have been disclosed in Chinese patents ZL 03153486.4 and ZL 03124061.5 respectively, but their applications mainly include molecular immobilization and related detection. Methods involving the isolation of mRNA

Method used

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  • Method for separating mRNA by using gold magnetism particles
  • Method for separating mRNA by using gold magnetism particles
  • Method for separating mRNA by using gold magnetism particles

Examples

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preparation example Construction

[0062] 1) Preparation of streptavidin-gold magnetic particles;

[0063] 1.1) Take 1mg of gold magnetic particles, equilibrate twice with 0.01MTris-HCl (pH8.0), add 200μg streptavidin, the reaction volume is 500μl, react in a shaker at 37°C, 180r / min for 20min, and react After completion, wash with 2×STE (pH8.0) buffer three times;

[0064] 1.2) Measure the OD value of the supernatant at 280nm before and after streptavidin immobilization and after washing with an ultraviolet-visible spectrophotometer, calculate the coupling efficiency and immobilization amount of streptavidin on the surface of gold magnetic particles, add 1ml containing 0.01%NaN 3 , 2×STE (pH8.0) buffer solution of 1% BSA, stored at 4°C for future use;

[0065] 2) Mix the biotin-labeled oligo(dT) probe with streptavidin-gold magnetic particles, and immobilize the biotinylated oligo(dT) probe on the surface of the magnetic particles through the combination of streptavidin-biotin , as a solid phase carrier for...

Embodiment 1

[0078] This example is the process of immobilizing streptavidin on the surface of gold magnetic particles in the present invention.

[0079] Take 1 mg of gold magnetic particles, equilibrate twice with 0.01M Tris-HCl (pH 8.0), add 200 μg streptavidin, and the reaction volume is 500 μl. React in a shaker at 37° C. and 180 r / min for 10 min. After the reaction is complete, wash with 2×STE (pH 8.0) buffer three times, 500 μl each time. Ultraviolet-visible spectrophotometer was used to measure the OD value of the supernatant at 280nm before and after streptavidin was immobilized and after washing, and the coupling efficiency and immobilized amount of streptavidin on the surface of gold magnetic particles were calculated. Add 1ml containing 0.01% NaN 3 , 1% BSA in 2×STE (pH 8.0) buffer, stored at 4°C for later use.

[0080] The UV absorption curves before and after the coupling of gold magnetic particles to streptavidin are as follows: figure 2 As shown, wherein curve 1 is the u...

Embodiment 2

[0082] This example is the process of immobilizing biotin-labeled oligonucleotide probes on the surface of streptavidin-gold magnetic particles in the present invention.

[0083] Take 250 μg streptavidin-gold magnetic particles, equilibrate three times with 2×STE buffer, magnetically separate, add 150 μl biotin-labeled oligonucleotide (dissolved in 2×STE, containing 862.5 pmol oligonucleotide), React in a shaker at 37°C and 120r / min for 10min. After the reaction, magnetically separate and wash thoroughly with 2×STE buffer. Measure the OD value of the supernatant before and after immobilization and after washing at 260nm, and calculate the biological Coupling efficiency and immobilization amount of prime-labeled oligonucleotides on the surface of gold magnetic particles.

[0084] The UV absorption curves before and after immobilization of biotin-labeled oligonucleotide probes on streptavidin-gold magnetic particles are as follows: image 3 As shown, curve 1 is the ultraviolet ...

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Abstract

The present invention relates to biotechnology, and is especially a technological scheme of magnetic metal particle process for separating out mRNA. The process includes the following steps: 1. preparing chain avidin-magnetic metal particle; 2. mixing biotin marked oligo(dT) probe and the chain avidin-magnetic metal particle to fix oligo(dT) probe with biotin onto the surface of magnetic metal particle through the combination of chain avidin and biotin, so as to form the solid carrier for reaction and separation; 3. hybridizing oligo(dT) probe and mRNA polyA specifically, magnetically separating and abandoning the supernatant; 4. washing magnetic metal particle with washing buffer solution to eliminate impurity; and 5. eluting mRNA with eluting buffer solution. The present invention has the advantages of low cost, simple operation and fast separation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating mRNA by using gold magnetic particles. Background technique [0002] Isolating mRNA from tissues or cells is a basic technique in the fields of molecular biology and genetic engineering. There is only about 10 pg of RNA in a typical mammalian cell, and mRNA only accounts for 1-5% of the total RNA. Commonly used mRNA separation methods are: (1) oligo(dT)-cellulose column chromatography, based on oligo(dT)-cellulose affinity chromatography column for PolyA + Specific adsorption of mRNA, followed by elution with elution buffer to obtain PolyA + mRNA. (2) oligo(dT)-cellulose liquid phase centrifugation method, that is, oligo(dT)-cellulose is directly added to the total RNA solution to make PolyA + The mRNA is combined with oligo(dT)-cellulose, and the oligo(dT)-cellulose / mRNA complex is collected by centrifugation, and the mRNA is separated by eluent, and then...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 崔亚丽陈超张战凤于桉
Owner XIAN GOLDMAG NANOBIOTECH
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