RNA affinity media and preparation method thereof

A medium and affinity technology, applied in the field of molecular biology, can solve problems such as the influence of RNA and its binding protein, change the spatial configuration of RNA molecules, etc., and achieve the effect of strong selectivity and easy preparation

Inactive Publication Date: 2011-06-22
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although RNA has a greater degree of freedom of movement in this type of medium, since the bifunctional reagent can still bind to it at all possible positions of the RNA molecule, it is possible to change the spatial configuration of the RNA molecule, so the role of RNA and its binding protein will still be limited. Affected

Method used

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  • RNA affinity media and preparation method thereof
  • RNA affinity media and preparation method thereof
  • RNA affinity media and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, preparation of target RNA with "tail"

[0036]1.1. The 3'-untranslated region of the mRNA of nuclear transcription factor C / EBPβ is cDNA with a length of 0.28kb. For the specific nucleotide sequence, see Liu Dingqian et al. (Liu Dingqian, Zhu Lihua, Chen Zhenzhen, Noda Liang, Guo Lihe, Li Zaiping, with The nucleotide sequence of a cDNA clone of anti-cancer gene activity, the literature of Biochemistry and Biophysics Acta 1991; 23 (3): 246-250), it is forward cloned into the multi-cloning position of plasmid pSP64 (Promega company) point. For the specific operation of cloning, please refer to Liu Dingqian et al. Recombinant plasmid pSP64 / 0.28. like figure 1 As shown, downstream of the multiple cloning site of the plasmid, the recombinant plasmid pSP64 / 0.28 was digested with PvuII to linearize the recombinant plasmid pSP64 / 0.28 to obtain a linear DNA containing the pSP promoter and 0.28kb cDNA.

[0037] 1.2. Use a kit (RiboMax large scale RNA production kit...

Embodiment 2

[0038] Embodiment 2, the treatment of glass frit carrier and aminosilanization

[0039] Ordinary glass powder (50-100 mesh) is washed with chloroform to remove oil stains, then fully washed with 5N NaOH and 5N HCl in turn, rinsed with a large amount of distilled water to remove residual HCl, and dried for later use.

[0040] Clean glass powder is soaked in 50% acetone solution of 3-aminopropyltriethoxysilane (purchased from Sigma Company), soaked for half an hour at room temperature, and then fully wash away residual 3-aminopropyltriethylsilane with acetone. Oxysilane, dried at 50°C for later use.

Embodiment 3

[0041] Embodiment 3, immobilizing DNA on the surface of aminosilanized glass frit carrier

[0042] 3.1, such as figure 2 As shown, the plasmid pSP65 (Promega) was digested and linearized with EcoRI, and the specific conditions were as follows:

[0043] Plasmid pSP65 20mg 2mL

[0044] 10 times concentration EcoRI buffer 250μL

[0045] 0.1% bovine serum albumin 250μL

[0046] EcoRI 1000 units (100 μL)

[0047] Incubate at 37°C overnight (12-16 hours).

[0048] The next day, take 2 μL of electrophoresis to check whether the digestion is complete; if the digestion is not complete, add 1000 units of EcoR I, continue to incubate for 4 hours, and check by electrophoresis to confirm that the digestion is complete.

[0049] 3.2. Extract the reaction solution with complete digestion with an equal volume of Tris-HCl (pH8) saturated phenol / chloroform (v / v 1:1) once, extract once with chloroform, take out the water phase, and add twice the volume of absolute ethanol , centrifuge at ...

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Abstract

The invention discloses RNA compatible medium and its manufacturing method used to separate and identify RNA singleness conjugated protein. It includes the following steps: fixing one end of DNA fragment of the plasmid pSP65 on glass dust of amino silane to form double chain DNA-glass dust; cloning target RNA gene or cDNA into another reciprocal clone site plasmid Psp64 to gain recombinant plasmid; gaining DNA fragment with extended nucleotide sequence by enzyme cutting; transcript RNA target sequence with RNA complementation fragment which is complemented with the plasmid pSP65 DNA fragment;heating the double chain DNA-glass dust and RNA target sequence together then cooling to make the RNA target sequence and signal chain DNA hybridize to form new RNA compatible medium which has the advantage of easy preparation, strong selectivity, repeatable utilization, etc.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an RNA affinity medium for separating and identifying RNA-specific binding proteins and a preparation method thereof. Background technique [0002] In the research work of molecular biology, the isolation and identification of RNA-specific binding proteins is an important means to study gene expression and regulation, and it is also an important method to study the molecular mechanism of disease occurrence and development. [0003] At present, there are many methods for isolating and identifying RNA-specific binding proteins. The principle is to simulate the cell microenvironment in a test tube, and contact the affinity medium with the target RNA with the cell protein solution in the artificially prepared binding buffer. To make the target RNA and its specific binding protein combine with each other, and then the binding protein is eluted and identified. In these met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07H21/00C07H1/06
Inventor 刘定干
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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