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Method for purifying RNA on preparative scale by means of HPLC

An alkylation and mobile phase technology, applied in DNA preparation, recombinant DNA technology, organic chemistry, etc., can solve complex and expensive gradient procedures and other problems, and achieve the effects of simple recovery, sensitive detection, and easy automatic operation

Active Publication Date: 2014-03-12
CUREVAC REAL ESTATE GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, this HPLC method involves the use of a relatively complex and expensive gradient program
Furthermore, this method is only capable of isolating and analyzing assay amounts of RNA up to 1000 ng (1 μg or 0.001 mg)

Method used

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  • Method for purifying RNA on preparative scale by means of HPLC
  • Method for purifying RNA on preparative scale by means of HPLC
  • Method for purifying RNA on preparative scale by means of HPLC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Utilize the HPLC method according to the present invention to purify 1.5 mg luciferase mRNA

[0089] Luciferase mRNA with a size of 1825 base pairs was used for isolation. A porous non-alkylated polystyrene / divinylbenzene (polystyrene divinylbenzene) matrix (commercially available from Polymer Laboratories) was used as the stationary phase. Its particle size is 8 μm and its pore size is The columns used were 5.0 cm in length and 7.5 mm in diameter. The temperature of the injector was 12°C, and the temperature of the HPLC separation, specifically, the temperature of the separation column was 78°C, that is, the operation was performed under completely denaturing conditions.

[0090] Separation was performed by the following gradient program:

[0091] Eluent A: 0.1M triethylammonium acetate, pH7

[0092] Eluent B: 0.1M triethylammonium acetate, pH7, containing 25vol.% acetonitrile

[0093] Eluent composition:

[0094] Start: 62% A and 38% B (1st to 3rd ...

Embodiment 2

[0104] Example 2: Use aperture as The stationary phase separates 200 μg of 2kb and 4kb RNA fragments

[0105] 200 μg of 2kb and 4kb RNA fragments were used for isolation. A porous non-alkylated polystyrene / divinylphenyl substrate (commonly available from Polymer Laboratories) was used as stationary phase. Its particle size is 8 μm and its pore size is The columns used were 2.5 cm long and 4.6 mm in diameter. The injector temperature was 12°C, and the HPLC separation temperature, specifically, the temperature of the separation column was 78°C, that is, the operation was performed under completely denaturing conditions.

[0106] Separation was performed using the following gradient program:

[0107] Eluent A: 0.1M triethylammonium acetate

[0108] Eluent B: 0.1M triethylammonium acetate / 25% acetonitrile

[0109] Eluent composition:

[0110] · Starting level: 62% A and 38% B (1st to 3rd minutes)

[0111] Separation range I: Gradient 38%-49.5% B (B increase 5.75% / min) ...

Embodiment 3

[0116] Example 3: Use aperture as The stationary phase separates 100 μg of 2kb and 4kb RNA fragments

[0117] 100 μg of 2kb and 4kb RNA fragments were used for isolation. A porous non-alkylated polystyrene / divinylphenyl substrate (commonly available from Polymer Laboratories) was used as stationary phase. Its particle size is 8 μm and its pore size is The columns used were 2.5 cm in length and 4.6 mm in diameter. The injector temperature was 12°C, and the HPLC separation temperature, specifically, the temperature of the separation column was 78°C, that is, the operation was performed under completely denaturing conditions.

[0118] Separation was performed using the following gradient program

[0119] Eluent A: 0.1M triethylammonium acetate

[0120] Eluent B: 0.1M triethylammonium acetate / 25% acetonitrile

[0121] Eluent composition:

[0122] · Starting level: 62% A and 38% B (1st to 3rd minute)

[0123] Separation range I: Gradient 38%-49.5% B (B increase 5.75% / min...

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Abstract

The application describes a method for the preparative purification of RNA, which method is distinguished in that the RNA is purified by means of HPLC using a porous reversed phase as the stationary phase. The use of the porous reversed phase in this HPLC method is also described.

Description

technical field [0001] The present invention relates to a method for the purification of RNA by HPLC on a preparative scale (preparative scale) and the use of a porous reversed phase as a stationary phase in this method. Background technique [0002] HPLC (short for "High Performance (High Pressure) Liquid Chromatography") is an established method for separating mixtures of substances that is widely used in biochemistry, analytical chemistry, and clinical chemistry. [0003] In the simplest case, an HPLC setup consists of a pump with an eluent reservoir (filled with mobile phase), a sample injection system, a separation column filled with a stationary phase, and a detector. Furthermore, a fraction collector can also be provided, with which individual fractions can be collected in each case after separation and can thus be used in further applications. [0004] RNA analysis using ion-pair reverse-phase HPLC is known from A. Azarani and K. H. Hecker (Nucleic Acids Research, v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12N15/10C07H21/00
Inventor 托马斯·克特雷尔弗洛里安·冯德米尔贝拉迪斯劳斯·赖德尔托尔斯藤·穆茨克
Owner CUREVAC REAL ESTATE GMBH
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