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Method for identifying target RNA (Ribonucleic Acid) of RNA binding protein through improved CLIP method

A technology for binding proteins and targets, which is applied in the fields of molecular biology and cell biology, can solve problems such as impact, and achieve the effects of overcoming steric hindrance, improving success rate, and reducing degradation

Inactive Publication Date: 2012-08-08
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] Aiming at the problem that CLIP uses RNase T1 or RNase A to do partial enzymolysis of RNA in the prior art, causing influence on downstream experiments, the present invention adopts micrococcal nuclease (micrococcal nuclease) to replace RNase A and RNaseT1, because micrococcal The cleavage activity of nucleases depends on Ca 2+ , after the enzyme digestion reaction, add buffer containing EGTA to chelate Ca in the solution 2+ , can completely inactivate micrococcal nuclease without affecting downstream experiments

Method used

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  • Method for identifying target RNA (Ribonucleic Acid) of RNA binding protein through improved CLIP method
  • Method for identifying target RNA (Ribonucleic Acid) of RNA binding protein through improved CLIP method
  • Method for identifying target RNA (Ribonucleic Acid) of RNA binding protein through improved CLIP method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Design and synthesis of RNA linker

[0032] The linker sequence used matches the UCSD Solexa sequencer. The sequence is 5'linker: 5'Biotin-AAU GAU ACG GCG ACC ACC GA 3'; 3'linker: 5'PO4-UCG UAU GCC GUC UUC UGC UUG-Puromycin. The biotin at the 5' end of the 5'linker and the puromycin at the 3' end of the 3'linker can block the end of the linker to prevent self-connection as described above. The reverse transcription primer is the reverse complementary DNA sequence of the 3' linker, namely: 5'CAA GCA GAA GAC GGC ATA CGA, the forward primer used in PCR is 5'AAT GAT ACG GCG ACC ACC GA, and the reverse primer is the reverse transcription primer.

Embodiment 2

[0033] Embodiment 2: Preparation of ultraviolet cross-linking and cell lysate

[0034] 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;

[0035] 2) Put the petri dish on ice, put it into the lower drawer of HL-2000 crosslinker (UVP), use 400mj / cm 2 The energy of 254nm ultraviolet light is irradiated for 1 to 1.5 minutes. Then take out the culture dish, pour off the PBS, add 15ml of PBS, and scrape off the cells with a cell scraper;

[0036] 3) Pipette 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 for 5 min...

Embodiment 3

[0042] Example 3: Co-immunoprecipitation

[0043] 1) Couple 300 μl of protein A magnetic beads (Protein A beads, Dynal beads, Invitrogen 100.02) to U2AF65 monoclonal antibody (MC3, Abcam Company). Aspirate the supernatant after bead precipitation;

[0044] 2) Add 900 μl of elution buffer (same as Example 2) to resuspend the magnetic beads, put them on the magnetic separation rack until the magnetic beads are precipitated, and repeat this three times;

[0045] 3) Add 700 μl of the cell lysate in Example 1 to the magnetic beads in 2), resuspend the magnetic beads until they are completely suspended in the cell lysate, place them on a rotary mixer, and mix at 4°C for one hour;

[0046] 4) Take out the supernatant with a pipette and keep 50 μl for Western detection of IP efficiency ( image 3 ), the rest of the supernatant was discarded;

[0047] 5) Add 900 μl of elution buffer to the magnetic beads bound to U2AF65 protein, elute twice as described above, then add high-salt was...

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Abstract

The invention discloses a method for identifying a target RNA (Ribonucleic Acid) of an RNA binding protein through an ultraviolet crosslinking-immune co-precipitation method. According to the method, micrococcalnuclease is used for performing incomplete enzymolysis instead of RNaseA and RNaseT1, and the micrococcalnuclease is completely inactivated by removing calcium ions (Ca<2+>) from a solution after enzymolysis is finished. Moreover, joints are arranged at the two ends of the target RNA in way of connecting 5'RNAlinker firstly and connecting 3'RNAlinker secondly after enzymolysis is finished. The micrococcalnuclease is used for clearing a part of RNAs, so that the degradation of the target RNA is effectively reduced; and due to the adoption of the scheme for connecting the 5'RNAlinker firstly, possible steric hindrance formed at a 3'end by a possibly-existing protein compound is overcome, and the successful rate of a CLIP technology is increased.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and relates to the identification of target RNA motifs bound by intracellular RNA binding proteins. More precisely, the present invention is an improvement and optimization of a published UV crosslinking-coimmunoprecipitation coupling experiment technique. Background technique [0002] RNA-binding proteins are a large class of very important proteins in organisms and have a wide range of biological functions. Many RNA-binding proteins have been found to be involved in numerous human diseases, such as autoimmune and neurological diseases. Understanding the roles of these RNA-binding proteins in normal and abnormal cells requires first identifying the location of the target RNAs they bind in vivo and the motifs of these target RNAs. In the past, the commonly used means of identifying these target RNAs was mainly through the phylogenetic evolution technique of in vitro exponentia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 张翼付向东吴同彬薛愿超邵长伟黄晨
Owner 武汉生命之美科技有限公司
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