A kind of rna pulldown method and kit

A technology of RNA probes and magnetic beads, applied in biological testing, material inspection products, measuring devices, etc., can solve the problems of nuclease contamination, low binding specificity between RNA probes and proteins, false positives, etc., and achieve Prevent contamination, save experimental cost, and improve specificity

Active Publication Date: 2018-03-20
GUANGZHOU BIOSENSE BIOSCI
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Problems solved by technology

[0006] The above-mentioned RNA pulldown method has obvious disadvantages: (1) the beads (agarose beads or magnetic beads) used in the experiment bind non-specifically to the protein, which may easily cause false positives in the experimental results; (2) RNA probes and protein The binding specificity is not high; (3) The experimental process will also be affected by nuclease contamination

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  • A kind of rna pulldown method and kit

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Embodiment 1

[0037] The present invention is illustrated by taking the pulldown effect of the lincRNA HOTAIR probe on the LSD1 protein as an example, and the probe is labeled with desthiobiotin 8-oxoG. The design and labeling methods of probes 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; the whole cell protein is incubated with magnetic beads-RNA probe, and the protein molecule can be specifically combined with the probe; after washing, the non-specific binding protein Molecule removal; finally, elution is performed with an eluent (for streptavidin) to obtain the target probe-protein complex, and then the protein type is identified by Western Blot. Specific steps are as follows:

[0038] S1. Magnetic bead pretreatment

[0039] ① Take 80 μL of streptavidin-containing magnetic beads and place them in an RNase-free EP tube...

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Abstract

The invention relates to an RNA pulldown method and a kit. The method includes the following steps of S1, pretreating magnetic beads by means of using BSA and a random oligonucleotide primer to enclose magnetic beads containing streptavidin; S2, preparing probe-magnetic bead complex; S3, extracting and pretreating whole cell proteins by means of adding ribonuclease into whole cell protein extraction samples for incubation, then adding the magnetic beads for incubation, and collecting supernatant; S4, performing pulldown; S5, collecting RNP. The method and the kit have the advantages that the BSA and the random oligonucleotide primer are used for enclosing the magnetic beads in advance, so that nonspecific binding of the magnetic beads with the proteins and RNA is lowered. Through systematic nuclease protection, pollution of nuclease is prevented, stability and reliability in tests are improved, repeatability is good, and test procedures are simplified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RNA pulldown method and kit for studying the interaction between RNA and protein. Background technique [0002] RNA-binding proteins (RBPs) play an important role in regulating gene expression at the post-transcriptional level, including splicing, polyadenylation and stability maintenance of mRNA precursors, transport of mRNA from the nucleus to the cytoplasm, mRNA positioning and translation etc. The regulatory role of RBPs in these processes is achieved by binding to specific sequences of newly synthesized or mature transcripts. At the same time, there are many RBPs that can recognize specific nucleotide sequences in RNA. Many methods have been established to study the interaction between RNA and protein, including EMSA, SELEX technology, RNA footprinting, RNA immunoprecipitation (RIP) and RNA pulldown technology. [0003] RNA pulldown technology utilizes end-labeled RNA to e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/573
CPCG01N33/573G01N33/68G01N2333/90245
Inventor 王晓香董先辉张娟曾健周剑
Owner GUANGZHOU BIOSENSE BIOSCI
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