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RNA (ribonucleic acid)purification chromatin separation technique

A separation technology and chromatin technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of low hybridization efficiency between probe sets and RNA, poor sensitivity, and low specificity of ChIRP technology. Achieve the effects of saving experimental costs, enhancing detection specificity, and improving hybridization efficiency

Pending Publication Date: 2016-12-07
GUANGZHOU BIOSENSE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing ChIRP technology has many disadvantages, such as magnetic beads will adsorb non-specific nucleic acids and proteins, and the hybridization efficiency between the probe set and RNA is not high, which leads to low specificity and poor sensitivity of ChIRP technology

Method used

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  • RNA (ribonucleic acid)purification chromatin separation technique
  • RNA (ribonucleic acid)purification chromatin separation technique
  • RNA (ribonucleic acid)purification chromatin separation technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] In this example, a ChIRP experiment was carried out for TINCR. TINCR is a long non-coding RNA, the species is human, the target gene is TGFBI, and the experimental material is human epidermal tissue.

[0054] A chromatin separation technique for RNA purification, comprising the following steps:

[0055] S1. Preparation of chromatin lysate

[0056] S11, cross-linked cells

[0057] All steps for crosslinking cells were performed at room temperature. 1v / v% formaldehyde PBS is prepared from 37% formaldehyde and PBS according to the volume ratio of 27:973, and it must be prepared and used immediately.

[0058] ①Suspend the cells with 1v / v% formaldehyde in PBS, pipette and mix well, shake and rotate at room temperature for 10 minutes, centrifuge at 1500-2000rpm for 5min and crosslink again;

[0059] ②End the cross-linking reaction, add 0.1mL of 1.25M glycine (about 0.01g) solution, shake and rotate at room temperature for 5 minutes; centrifuge at 2000RCF for 5 minutes, re...

Embodiment 2

[0102] In this example, a ChIRP experiment was carried out for FENDRR. FENDRR is a long non-coding RNA, the species is human, the target gene is NIPA1, and the experimental material is hLF cells.

[0103] A chromatin separation technique for RNA purification, comprising the following steps:

[0104] S1. Preparation of chromatin lysate

[0105] The steps described in Example 1 are the same.

[0106] S2, magnetic bead pretreatment

[0107] The steps described in Example 1 are the same.

[0108] S3. Preparation of probe-magnetic bead complex

[0109] S31. Probe pretreatment

[0110] In this embodiment, a plurality of probes are designed for lncRNA to form a probe group. The probe groups of the experimental group are Even group and Odd group, each of 5 probes in Even group and Odd group, and the blank control group (NC group) contains 5 probes. strip probes (see Table 1). Before pretreatment, the probe groups of the experimental group and the blank control group were config...

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Abstract

The invention relates to an RNA (ribonucleic acid) purification chromatin separation technique. The technique includes the following steps: binding probes with magnetic beads prior to hybridizing with chromatin lysis solution, and then performing washing for multiple times via gradient temperature. The magnetic beads are closed with BSA and random oligonucleotide primers in advance, and accordingly nonspecific binding of the magnetic beads with proteins and RNA is reduced. The magnetic beads are incubated with the probes firstly prior to being incubated with the proteins after excessive probes are removed, gradient temperature washing is performed after hybridization to assure that the magnetic beads fully bind with the probes and bind with target RNA, and hybridization efficiency is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chromatin separation technique for RNA purification. 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. As a new biological macromolecule, lncRNA plays an important role in the occurrence and development of human diseases. Therefore, it is necessary to study the molecular mechanism of lncRNA to provide technical support for good diagnosis and treatment of diseases. [0003] In 2011, Professor Howard Chang of Stanford University developed the chromatin isolation technology (chromatin isolation by RNA purification, ChIRP) for RNA purification. ChIRP technology is a genome-wide method to identify chromatin and protein interactions boun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6816C12Q2563/143C12Q2563/149C12Q2527/125
Inventor 王晓香董先辉张娟曾健周剑
Owner GUANGZHOU BIOSENSE BIOSCI
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