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Method for rapidly and completely obtaining mRNA ribosome nascent-chain complex by using molecular sieve spin column and application of method

A spin column and molecular sieve technology, applied in the field of life sciences, can solve the problems of shortening the experiment time, reducing the cost of the experiment, and less experiment steps, and achieve the effects of shortening the experiment time, taking a long time to solve, and simple operation

Pending Publication Date: 2019-07-12
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Utilizing the direct penetration characteristics of molecular sieve ultra-large molecular weight substances, combined with low-speed centrifugation as a purification method, no expensive instruments such as ultracentrifuges and chromatography systems are required, only ordinary low-speed centrifuges are required, the experimental steps are few, the operation is simple, and the probability of contamination by RNase Small size, greatly shortening the experimental time and greatly reducing the experimental cost, and at the same time, it can obtain RNC-mRNA sequencing data with the same quality as the sucrose density ultracentrifugation method, or even better, which can effectively overcome the current method's high cost, complicated operation, and low throughput. technical flaw

Method used

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  • Method for rapidly and completely obtaining mRNA ribosome nascent-chain complex by using molecular sieve spin column and application of method
  • Method for rapidly and completely obtaining mRNA ribosome nascent-chain complex by using molecular sieve spin column and application of method
  • Method for rapidly and completely obtaining mRNA ribosome nascent-chain complex by using molecular sieve spin column and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Use a molecular sieve spin column to quickly and completely obtain the mRNA ribosome nascent peptide chain complex in animal tissue (BALB / C mouse kidney), the specific steps are as follows:

[0058] (1) Take out the molecular sieve spin column (MicroSpin S-400 HR-GE Healthcare), use 3ml of RNase-free column equilibration buffer (20mM, HEPES-KOH solution of pH7.4, 15mM MgCl 2 , 200mM KCl) for column equilibration, and then placed in a 4°C refrigerator until the column packing settled overnight.

[0059] (2) 6-week-old BALB / C mice (Guangdong Provincial Medical Animal Experiment Center) were dissected to obtain kidney organs, the blood was quickly cleaned with DPBS solution, and put into liquid nitrogen for quick freezing.

[0060] (3) Put the mouse kidney into a grinding tube, and grind it with a tissue grinder.

[0061] (4) Add 1ml of cell lysate to the grinding tube, the composition of the cell lysate is as follows: 20mM HEPES-KOH solution with pH7.4, 15mM MgCl 2 , 20...

Embodiment 2

[0077] Use a molecular sieve spin column to quickly and completely obtain the mRNA ribosome nascent peptide chain complex in human non-small cell lung cancer cell A549 (Cell Bank, Chinese Academy of Sciences), and the steps are as follows:

[0078] (1) Take out the molecular sieve spin column (MicroSpin S-400 HR-GE Healthcare), use 3ml of column equilibration buffer (20mM, HEPES-KOH solution of pH7.4, 15mM MgCl 2 , 200mM KCl) for column equilibration, and then placed in a 4°C refrigerator until the column packing settled overnight.

[0079] (2) Add 1ml of cell lysate to the cell culture flask (10 million A549 cells), the composition of the cell lysate is as follows: 20mM HEPES-KOH solution with pH7.4, 15mM MgCl 2 , 200 mM KCl, 100 μg / ml cycloheximide, 2 mM DTT, 1% (v / v) Triton X-100; lyse for 25 min to obtain a cell lysate.

[0080] (3) The cell lysate was centrifuged at 17000g for 5min to remove large fragments, and the supernatant was centrifuged at 17000g for 15min to remo...

Embodiment 3

[0095] Use the molecular sieve spin column to quickly and completely obtain the mRNA ribosome nascent peptide chain complex in the microorganism (Escherichia coli K-12BW25113, BioVector China Plasmid Vector Strain Cell Protein Antibody Gene Collection Center), the steps are as follows:

[0096] (1) Take out the molecular sieve spin column (MicroSpin S-400 HR-GE Healthcare), use 3ml of column equilibration buffer (10mM, Tris of pH7.8, 50mM NH 4 Cl, 0.01M MgCl 2 ) for column equilibration, and then placed in a 4°C refrigerator until the column packing settled overnight.

[0097](2) Escherichia coli BW25113 glycerol species were streaked on LB plates and cultured overnight.

[0098] (3) Pick a single colony on the plate, inoculate in 3ml LB liquid medium, and activate overnight at 37°C and 200rpm.

[0099] (4) Inoculate the activated seed solution with an inoculation amount of 1% by volume, inoculate it in 50ml LB liquid medium, and cultivate it at 37°C and 200rpm until the OD ...

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Abstract

The invention provides a method for rapidly and completely obtaining an mRNA rribosome nascent-chain complex by using a molecular sieve spin column and application of the method. The method comprisesthe steps that a lysate is utilized for conducting pyrolysis on a biological sample to obtain a lysate product; debris in the lysate is removed through differential centrifugation to obtain a supernatant after pyrolysis; the supernatant after pyrolysis is purified by using the molecular sieve spin column to obtain the high-purity mRNA rribosome nascent-chain complex. The method has the advantagesthat a valuable instrument ultracentrifuge is not needed, the operation is simple, few steps are needed, the probability of being contaminated by RNase is low, the experimental repeatability is high,the step of purification by using the molecular sieve spin column takes only about 10 minutes, the experimental time is greatly shortened, large-scale operation can be conducted, the limitations of long time consumption, large operation difficulty, low throughput and the like of a sucrose density ultracentrifugation method are overcome, high-quality RNC-mRNA sequencing data can be obtained, and animportant role is played in promoting the popularization of an RNC technology.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a method for rapidly and completely obtaining mRNA ribosome nascent peptide chain complexes by using a molecular sieve spin column and an application thereof. Background technique [0002] Translational regulation is an important regulatory level in living organisms. Translational regulation accounts for more than 50% of the total regulation of living organisms. The expression of genes in living organisms does not mean that they enter the translation process to synthesize proteins. Therefore, it is extremely important to study the translation status of living organisms. When the cell is translating, the ribosome binds to the mRNA chain and moves to synthesize protein. This complex composed of complete mRNA, ribosome and nascent peptide chain is the mRNA ribosome Nascent-chain Complex (RNC) , only when intact and undegraded RNCs are extracted, can the qualitative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869
CPCC12N15/1006C12Q1/6806C12Q1/6869
Inventor 卢小龙张弓
Owner JINAN UNIVERSITY
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