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A method for detecting the expression of cellular RNA

A cell and N-terminal technology, applied in the field of genetic engineering, can solve problems such as low signal-to-noise ratio, cell fixation, and inapplicability, and achieve the effect of low signal-to-noise ratio and improved signal-to-noise ratio

Active Publication Date: 2022-06-07
XUZHOU MEDICAL UNIV
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Problems solved by technology

[0007] Aiming at the deficiencies of the existing technology, this application solves the existing problems that cells need to be fixed and is not suitable for live cell imaging; it combines the technical advantages of bimolecular fluorescence complementary technology and RNA binding protein-fluorescent molecule technology, aiming at Provide a method for detecting intracellular RNA molecules with a low signal-to-noise ratio. This method has the advantages of high efficiency, simplicity, economy and accuracy

Method used

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Embodiment 1

[0025] The method for detecting the expression of cellular RNA is based on the principle of bimolecular fluorescence complementation. Different RNA-binding proteins or domains are fused at the N-terminus and C-terminus of the fluorescent molecule to express different RNA binding proteins or domains. The RNA binding to be detected, and the two exogenously expressed RNAs contain RNA binding protein binding sequences, which will emit fluorescence when the two fusion expressed RNA binding proteins bind to them.

[0026] There is a flexible peptide sequence between the RNA binding protein and the fusion protein of the N-terminal and C-terminal ends of the fluorescent molecule, and the flexible peptide sequence is GGGGS in SEQ ID NO.1 and GGGGS in SEQ ID NO.2.

[0027] The fusion protein of the RNA binding protein / domain and the N-terminus or C-terminus of the fluorescent molecule constructs a eukaryotic expression vector, which is directly transfected into cells by virus or plasmid ...

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Abstract

This application discloses a method for detecting cellular RNA expression. According to the principle of bimolecular fluorescence complementation, different RNA binding proteins or structural domains are fused at the N-terminal and C-terminal of the fluorescent molecule, and two exogenously expressed RNA aptamers pass through The principle of complementary base pairing is combined with the RNA to be detected, and the two RNAs expressed exogenously contain RNA binding protein binding sequences, and when the two fusion expressed different RNA binding proteins bind to them, they will emit fluorescence.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for detecting cellular RNA expression. Background technique [0002] RNA directly regulates a large number of cellular processes, and observing RNA can bring researchers many new insights. The presence of RNA in specific cells and in specific sites in those cells can reveal the mechanisms underlying the differences. Robust methods are needed to fluorescently label and track RNA in living cells. [0003] RNA imaging can also answer some of the questions raised by gene expression profiling studies. Scientists have traced some transcripts for further study. RNA in situ hybridization can show how a gene is expressed across a population of cells, which pairs of transcripts increase and decrease together in the same cell, whether a transcript is localized in a specific cell type or in a particular cell like dendrites Inside the area. In situ hybridization can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12N15/115
CPCG01N21/6458G01N21/6428C12N15/115G01N2021/6439C12N2310/16
Inventor 李丹花黄亚琦刘清华李燕强
Owner XUZHOU MEDICAL UNIV
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