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Method of separating whole length, complete messenger RNA from total RNA

A technology of messenger ribonucleic acid and total ribonucleic acid, applied in chemical instruments and methods, sugar derivatives, sugar derivatives, etc., can solve the problems of low mRNA isolation rate, high price, and failure of mRNA isolation, and achieve repeatability Good, low cost, and the effect of improving purity

Inactive Publication Date: 2005-02-16
JUNXUAN BIOLOGICAL TECH SHENZHEN CITY +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0011] Although the kit brings a lot of convenience to the mRNA isolation work, there are still shortcomings: (1) The yield of mRNA isolation is not high; (2) The price is expensive, which is difficult for laboratories that need to isolate mRNA in large quantities of
[0012] In addition, currently commonly used total RNA isolation methods, such as TRIZOL method and "one-step method" (ChomzynskiP., SacchiN..Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction.Anal.Biochem., 1987, 162:156-159.), etc., there is often residual endogenous RNase activity in the isolated total RNA, which often leads to the failure of mRNA isolation

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  • Method of separating whole length, complete messenger RNA from total RNA
  • Method of separating whole length, complete messenger RNA from total RNA

Examples

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Embodiment 1

[0068] Example 1: Isolation and purification of mRNA from total RNA isolated from mouse tissues.

[0069] A Required reagents and materials

[0070] 1. Total RNA of 8 kinds of mouse tissues (brain, heart, liver, lung, spleen, kidney, testis, skeletal muscle) (separated by "one-step method").

[0071] 2. RNase-free water (with 1g / L SDS added).

[0072] 3. Adsorption buffer: 20mmol / L Tris-HCl and 2mmol / L EDTA, 1mol / L NaCl and 1g / L SDS at pH 7.5.

[0073] 4. Equilibration buffer: 10mmol / L Tris-HCl and 1mmol / L EDTA, 0.5mol / L NaCl and 1g / L SDS at pH 7.5.

[0074] 5. Wash buffer: 10 mmol / L Tris-HCl and 1 mmol / L EDTA and 0.15 mol / L NaCl and 1 g / L SDS at pH 7.5.

[0075] 6. Elution buffer: 10 mmol / L Tris-HCl and 1 mmol / L EDTA at pH 7.5.

[0076] Note: Except for Tris, all the above solutions need to be treated with DEPC (diethylpyrocarbonate) to 1g / L overnight, and then autoclaved. Tris solution needs to be prepared with RNase-free Tris, water and container, and then autoclaved. ...

Embodiment 2

[0094] Example 2: Isolation and purification of mRNA from total RNA isolated from human tissue.

[0095] A Required reagents and materials

[0096] 1. Total RNA (separated with TRIZOL reagent) of 8 human tissues (brain, heart, liver, lung, spleen, stomach, testis, and skeletal muscle).

[0097] 2. RNase-free water (with 1g / L SDS added).

[0098] 3. Adsorption buffer: 20mmol / L Tris-HCl and 2mmol / L EDTA, 1mol / L NaCl and 1g / L SDS at pH 7.5.

[0099] 4. Equilibration buffer: 10mmol / L Tris-HCl and 1mmol / L EDTA, 0.5mol / L NaCl and 1g / L SDS at pH 7.5.

[0100] 5. Wash buffer: 10 mmol / L Tris-HCl and 1 mmol / L EDTA and 0.15 mol / L NaCl and 1 g / L SDS at pH 7.5.

[0101] 6. Elution buffer: 10 mmol / L Tris-HCl and 1 mmol / L EDTA at pH 7.5.

[0102] Note: Except for Tris, all the above solutions need to be treated with DEPC (diethylpyrocarbonate) to 1g / L overnight, and then autoclaved. Tris solution needs to be prepared with RNase-free Tris, water and container, and then autoclaved.

[01...

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Abstract

A process for separating the whole-length complete messanger RNA (mRNA) from total RNA features that the sodium lauryl sulfate (or sulfonate) is added to the solution of total RNA or the solution used to separate mRNA for preventing the degradation of RNase to mRNA and two kinds of buffer liquid are used for washing to remove other RNAs other than mRNA and deactive RNase.

Description

technical field [0001] The present invention relates to a method for efficiently isolating full-length, intact messenger ribonucleic acid from total ribonucleic acid. technical background [0002] In the research of molecular biology and molecular genetics, although many analytical studies involving ribonucleic acid (RNA) can be done with total RNA samples, but in the construction of reverse transcription deoxyribonucleic acid (cDNA) library, detection of rare messenger ribose Nucleic acid (mRNA), S 1 High-quality full-length, intact mRNA samples must be used in studies such as nuclease mapping and in vitro translation. Therefore, the separation and purification of mRNA is one of the basic methods that must be mastered in the research of molecular biology. [0003] In the total RNA of eukaryotic cells, ribosomal ribonucleic acid (rRNA), transfer ribonucleic acid (tRNA) and the like account for more than 95%, while mRNA only accounts for 1-5%. Since most eukaryotic mRNAs h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H1/06C07H21/00C07H21/02
Inventor 叶志华梁培华郭俊张向阳丁野青丁达明梁建庆姚志海杜纲国高冬梅
Owner JUNXUAN BIOLOGICAL TECH SHENZHEN CITY
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