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Improved method for identifying target RNA sequences of RNA-binding protein in cell sample

A technology that binds proteins and targets, applied in the biological field, can solve problems such as isotope hazards, cumbersome steps, and complicated processes

Inactive Publication Date: 2018-02-09
武汉生命之美科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The existing Clip-seq technology process is complicated, the steps are cumbersome, and special equipment is required
Also requires the use of radioactive elements 32 The P labeling method indicates the position of the protein-RNA complex. Isotopes are harmful to the human body. The country has strict management on the use of isotopes. Ordinary laboratories are not qualified to apply for isotopes.

Method used

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  • Improved method for identifying target RNA sequences of RNA-binding protein in cell sample
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  • Improved method for identifying target RNA sequences of RNA-binding protein in cell sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] [Example 1] Design and synthesis of RNA linker

[0054] The linker sequence used matches illumina's Nex-500 sequencer:

[0055] 3'linker: 5'-PO 4 ATC TGG AAT TCT CGG GTG CCA AGG-Puromycin 3' (Seq ID NO: 1);

[0056] 5'linker: 5'Biotin-TGG AAT TCT CGG GTG CCA AGG 3'(Seq ID NO:2);

[0057] The puromycin at the 3' end of the 3'linker and the biotin at the 5' end of the 5'linker block the linker end to prevent head-to-tail self-connection.

[0058] The reverse transcription primer is the reverse complementary DNA sequence of the 3' linker:

[0059] 5'CCT TGG CAC CCG AGA ATT C (Seq ID NO:3),

[0060] Forward primers used in PCR:

[0061] 5'AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA (Seq ID NO:4);

[0062] The reverse primer is the reverse transcription primer.

Embodiment 2

[0063] [Example 2] Preparation of ultraviolet crosslinking and cell lysate

[0064] 1) Use a 150mm cell culture dish to culture HeLa cells, use DMEM (Invitrogen) as the medium, discard the medium when the cell abundance reaches over 80%, rinse twice with 1xPBS buffer, then add an appropriate amount of PBS buffer to cover cell surface;

[0065] 2) Put the petri dish on ice, put it into the drawer at the lower part of HL-2000 crosslinking instrument (MVP), make 400mj / cm 2 Energy of 254nm ultraviolet radiation 1 time. Then take out the culture dish, pour off the PBS, add 15ml of PBS, and scrape off the cells with a cell scraper;

[0066] 3) Use a pipette to suck the PBS suspension of all cells into a 15ml centrifuge tube, centrifuge at 4000rpm at 4°C for 5 minutes, and remove the supernatant. Add 1.5ml PBS, blow up the precipitate with a pipette, transfer to a clean 1.5ml centrifuge tube (DEPC treatment or use imported RNase free centrifuge tube), centrifuge again at 4000rpm f...

Embodiment 3

[0072] [Example 3] Fragmentation of RNA molecules

[0073]When performing a RIP experiment on a protein for the first time, it is necessary to explore the optimal concentration of MicrococalNμclease enzyme (MNase) (Fermentas company) so that the RNA tag is at least 20 nucleotides (20 nucleotides are MCSC Genome Browser for genome comparison (minimum length), the average length is preferably 30-60 nucleotides.

[0074] 1) Add Micrococal Nμclease enzyme and 10X MNase buffer according to the explored concentration, put it on the thermomixer for 10 minutes, 1000rpm, and mix for 15 seconds every 3 minutes;

[0075] 2) Add EDTA+EGTA solution to chelate Ca2+ in the system to terminate the reaction.

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Abstract

The invention relates to the field of biotechnology, and provides improved identification for target RNA sequences bound to RNA-binding protein in cells. Through UV crosslinking-immunopricipitation, target RNA of RNA-binding protein is obtained, micrococcal nuclease is adopted for carrying out incomplete enzymolysis on the target RNA after ultraviolet crosslinking, after enzymolysis, a chelating agent capable of removing Ca<2+> is adopted for inactivating the enzyme, the 3' terminal of the target RNA obtained through the method is phosphorylated, the phosphorylated 3' terminal is ligated witha 3' RNA linker, then, the ligation product is phosphorylated, the phosphorylated ligation product is separated, the separated phosphorylated ligation product is recycled, then the 3' terminal is ligated with a 5' RNA linker, RT-PCR amplification is carried out, so that a cDNA library corresponding to the target RNA is obtained. With the technical scheme provided by the invention, the experiment flow is simplified, the step of isotope labelling is eliminated, due to the adding of an IgG antibody negative control sample, the influences of non-specific binding and background on the experimentalresult are eliminated, and the data result with the quality equal to that of Clip-seq is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the identification of target RNA sequences bound by intracellular RNA binding proteins. Background technique [0002] RNA is an unstable biomacromolecule, and most RNAs need to be combined with specific RNA-binding proteins to form RNA / protein complexes in order to exist stably in cells; not only that, the dynamics between RNA and RNA-binding proteins The association runs through and is accompanied by the entire life cycle of RNA transcription synthesis, processing and modification, intracellular transport and localization, function and degradation. [0003] In view of this, the use of RNA-binding proteins to isolate or identify functional RNA molecules is an indispensable research method in the field of RNA research. In the past, the common means of identifying these target RNAs was mainly through the phylogenetic evolution technique of in vitro exponential enrichment of ligands (S...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1013C40B50/06
Inventor 张翼薛亚强张宇陈栋
Owner 武汉生命之美科技有限公司
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