Preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid)

A magnetic microsphere and reaction technology, applied in the biological field, can solve the problems of column chromatography, expensive, ineffective, etc., and achieve the effect of rapid separation

Pending Publication Date: 2022-04-08
SUZHOU KNOWLEDGE & BENEFIT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the TFF method is to filter impurities according to the different molecular weight cut-offs of the filter membranes. The equipment required is complex and expensive, and the filter membranes used as consumables have various specifications, and the replacement is cumbersome; the Oligo dT modified cellulose chromatography column method is based on Oligo dT will specifically bind to the poly(A) tail at ...

Method used

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  • Preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid)
  • Preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid)
  • Preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid)

Examples

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Embodiment 1

[0025] A preparation method for magnetic microspheres for purifying mRNA, comprising the steps of getting rigid polystyrene magnetic microspheres (knowledge benefits) with epoxy active groups on the surface brand), washed 5 times with 0.05M phosphate buffer solution with pH=7.4, added Oligo dT according to the ratio of 5nmol / mL polystyrene magnetic microspheres, mixed well at 25°C, reacted for 5 hours, and the reaction ended Finally, wash the magnetic microspheres with phosphate buffer solution of 0.05M pH=7.4 to wash away unreacted Oligo dT; add 10% BSA, mix well at 25°C, and react for 5 hours. After the reaction, The magnetic microspheres were washed with 0.1M pH=8.0 Tris-HCl buffer solution, and then the prepared Oligo dT magnetic microspheres were added to 0.01M pH=7.4 PBS buffer solution for use.

[0026] The specific purification method of the magnetic microspheres used for purifying mRNA prepared above is as follows: the total RNA obtained from cells is dissolved in RN...

Embodiment 2

[0028] A preparation method for magnetic microspheres for purifying mRNA, comprising the steps of getting rigid polystyrene magnetic microspheres (knowledge benefits) with benzenesulfonyl active groups on the surface brand), washed 3 times with 0.1M carbonate buffer solution with pH=8.4, added Oligo dT according to the ratio of 1nmol / mL polystyrene magnetic microspheres, mixed well at 25°C, and reacted for 3 hours. After the end, wash the magnetic microspheres with 0.1M carbonate buffer solution with pH=8.4 to wash away unreacted Oligo dT; add blocking reagent, mix well at 25°C, and react for 3 hours. , wash the magnetic microspheres with citric acid buffer solution of 0.1MpH=4.5, and then add the prepared Oligo dT magnetic microspheres into PBS buffer solution of 0.01MpH=7.4 for use.

[0029] The specific purification method of the magnetic microspheres used for purifying mRNA prepared above is as follows: the total RNA obtained from cells is dissolved in RNase Free (RNase F...

Embodiment 3

[0031] A preparation method for magnetic microspheres for purifying mRNA, comprising the steps of getting rigid polystyrene magnetic microspheres (knowledge benefits) with active ester (NHS) active groups on the surface brand), washed 3 times with 0.1M borax buffer solution with pH=8.4, added Oligo dT according to the ratio of 4nmol / mL polystyrene magnetic microspheres, mixed well at 4°C, and reacted for 17 hours. , using 0.1M borax buffer solution with pH=8.4 to wash the magnetic microspheres to wash away unreacted Oligo dT; add blocking reagent, mix well at 37°C, and react for 2 hours. After the reaction, wash with 0.05M The magnetic microspheres were washed with carbonate buffer solution of pH=9.0, and then the prepared Oligo dT magnetic microspheres were added to 0.01M PBS buffer solution of pH=7.4 for use.

[0032]The specific purification method of the magnetic microspheres used for purifying mRNA prepared above is as follows: the total RNA obtained from cells is dissol...

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Abstract

The invention discloses a preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid). The preparation method of the magnetic microspheres for purifying mRNA comprises the following steps: S1) washing the magnetic microspheres with active groups on the surfaces by using a coupling buffer solution; s2) Oligo dT is added to react with the magnetic microspheres, and then a coupling buffer solution is used for cleaning; and S3) adding a blocking reagent for reaction, and after the reaction is finished, cleaning with a coupling buffer solution to prepare the magnetic microspheres for purifying mRNA. The surface of the magnetic microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the magnetic microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the magnetic microsphere, so that the magnetic microsphere has mRNA separation and purification capabilities, and because the magnetic microsphere has magnetism, in the presence of a magnetic field, the mRNA separation and purification capability of the magnetic microsphere is greatly improved. The magnetic microspheres can quickly move towards one side of a magnetic field, so that mRNA can be quickly separated and purified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of magnetic microspheres for purifying mRNA. Background technique [0002] In eukaryotes, mature mRNA molecules are transferred from the nucleus to the cytoplasm and used as a direct template for protein synthesis. Therefore, mRNA is of great significance in the fields of molecular biology and vaccine preparation. [0003] A typical eukaryotic cell contains very little mRNA. Therefore, it is very challenging to isolate and purify mRNA. There are two traditional methods for isolating and purifying mRNA: 1. Tangential Flow Filtration (TFF); 2. Oligo dT modified cellulose chromatography column method. Among them, the TFF method is to filter impurities according to the different molecular weight cut-offs of the filter membranes. The required equipment is complicated and expensive, and the filter membranes used as consumables have various specificat...

Claims

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Application Information

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IPC IPC(8): B01J20/26B01J20/28B01J20/30C12N15/10
Inventor 阳承利李雪
Owner SUZHOU KNOWLEDGE & BENEFIT TECH CO LTD
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