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RNA (Ribonucleic Acid) antisense purification method

A purification method and antisense technology, applied in the field of RNA antisense purification, can solve the problems of low product recovery efficiency, poor sensitivity and low efficiency, and achieve the effects of reducing non-specific adsorption, improving detection specificity and high sensitivity

Inactive Publication Date: 2017-05-31
GUANGZHOU BIOSENSE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing RAP technology has the disadvantages of low specificity and poor sensitivity due to problems such as magnetic beads will adsorb non-specific nucleic acids and proteins, the hybridization efficiency of probe sets and RNA is not high, and the recovery efficiency of nucleic acid and protein products is not high.

Method used

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  • RNA (Ribonucleic Acid) antisense purification method

Examples

Experimental program
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Embodiment 1

[0034] CTNNB1 is mRNA encoding catenin beta 1 protein, and its species is human. The RAP experiment was carried out for CTNNB1 mRNA, and the target gene was hsa-miR-214. The design of the probe and the method of labeling with biotin are well known to those skilled in the art, and will not be repeated here. Streptavidin coupled to magnetic beads (BeaverBeads TM Streptavidin, Beaver Biotechnology Co., Ltd.) and affinity binding with dethiobiotin; RNA was incubated with magnetic beads-RNA probe, and non-specifically bound RNA could be removed after washing; finally, the eluent (for strand Mycoavidin) for elution, RNA type can be verified after purification. Specific steps are as follows:

[0035] S1. Cell cross-linking, lysis and genomic DNA digestion to obtain RNA samples

[0036] S11, culturing and collecting cells

[0037] In this example, cCL-RAP cell culture or PAR-CL cell culture was used. A 15cm cell culture dish was used for culture, cCL-RAP cells were cultured in ...

Embodiment 2

[0066] SENP1 is mRNA encoding SUMO1 / sentrin specific peptidase 1 protein, and its species is human. The RAP experiment was carried out for SENP1mRNA, and the target gene was lincRNA-p21.

[0067] S1. Cross-linking and lysis of cells and digestion of genomic DNA to obtain RNA samples. Concrete method is identical with embodiment 1.

[0068] S2. Preparation of the probe set solution: the specific method for preparing the probe set solution in this example is the same as that in Example 1, except that the sequence of the probe set used in the experimental group is different.

[0069] In this example, the probe set of the experimental group contains 10 probes, and the probe set of the NC group contains 3 probes (the same as in Example 1). The specific sequence is shown in Table 3.

[0070] Table 1. Probe sequences for experimental groups

[0071]

[0072] S3, magnetic bead pretreatment: the same as in Example 1.

[0073] S4. Preparation of the probe set-magnetic bead compl...

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Abstract

The invention relates to an RNA (Ribonucleic Acid) antisense purification method. The RNA antisense purification method comprises the following steps: S1, cross linking and pyrolysis of cells and digestion of genome DNA (Deoxyribonucleic Acid), thus obtaining an RNA sample; S2, preparation of a probe group solution; S3, pre-treatment of magnetic beads; S4, preparation of a probe group-magnetic bead compound; S5, antisense purification; S6, elution of RNA; S7, purification of the RNA. According to the RNA antisense purification method disclosed by the invention, the magnetic beads are sealed by using BSA (Bull Serum Albumin) and random oligonucleotide primer in advance, so that non-specific adsorption of the magnetic beads is reduced; the magnetic beads are in incubation with probes at first and then is in incubation with the RNA after residual probes are removed, non-specific combination of streptavidin with protein, nucleic acid and the like is sealed by excessive probes, the detection specificity is increased, and the sensitivity is high; multiple washing under a gradient temperature is carried out after hybridization, so that the magnetic beads are enabled to be fully combined with the probes and to be combined with a target RNA, and the hybridization efficiency is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RNA antisense purification method. Background technique [0002] Long non-coding RNA (Long non-coding RNA, lncRNA) is a transcript of more than two hundred nucleotides, most of which are located in the nucleus. Although lncRNAs do not encode any protein, their expression is still specific in different tissues and developmental stages, which indicates that lncRNAs have important biological significance. With the help of sequencing technology, thousands of lncRNAs have been discovered, but the biological functions of these lncRNAs are still a mystery. [0003] In 2013, Professor Jesse M. Engreitz developed the RNA antisense purification (RNA antisense Purification, RAP) technology, which can identify the interaction of RNA with RNA and related DNA and protein at the post-transcriptional level. The principle is as follows: First, cells are fixed with ultraviolet cross-linking to ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q1/6806C12Q2563/143C12Q2563/149
Inventor 王晓香董先辉张娟曾健周剑
Owner GUANGZHOU BIOSENSE BIOSCI
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