Novel method for screening microRNA (Ribose Nucleic Acid) target genes at high flux

A target gene, high-throughput technology, applied in the field of high-throughput screening of microRNA target genes, can solve the problem of false positives of target genes, and achieve the effect of simple operation, low cost, and avoiding false positives

Inactive Publication Date: 2011-07-06
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Application Information

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Problems solved by technology

However, these methods often speculate the target genes of microRNAs by detecting changes in the expression profile of the entire genome, and because some gene changes are not caused by the direct action of microRNAs, this may cause "false positives" in the screened target genes

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  • Novel method for screening microRNA (Ribose Nucleic Acid) target genes at high flux
  • Novel method for screening microRNA (Ribose Nucleic Acid) target genes at high flux
  • Novel method for screening microRNA (Ribose Nucleic Acid) target genes at high flux

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 experimental material and method

[0038] 1. Design and synthesis of microRNA mimics

[0039] The microRNA mimic is a structural double-stranded RNA artificially synthesized by simulating the hairpin-shaped precursor in the maturation process of endogenous microRNA. The antisense strand is reverse complementary to 19 nucleotides from the 5' end of the sense strand and has two bases each overhanging the 3' ends of the sense and antisense strands. The synthesis of microRNA mimics was done by Genepharma. In this experiment, two representative miRNAs were selected for subsequent experiments, and the oligonucleotide sequences involved in the experiment are as described in Table 1:

[0040] Table 1: Oligonucleotide sequences involved in the experiments

[0041] SEQ

ID

NO:

sequence name

sequence

1

miR-7 sequence

5'-UGGAAGACUAGUGAUUUUGUU

GU-3'

2

miR-7 mimetic

positive sequence

...

Embodiment 2

[0065] Embodiment 2 test result and main conclusion

[0066] Using the new technology of biotin-modified microRNA mimics for high-throughput screening of microRNA target genes, we randomly selected two microRNAs (miR-7 and miR-122) to design and synthesize microRNA double-stranded mimics. We first transfected target cells with FAM-modified microRNA mimics, and imaged by laser confocal ( figure 2 ) and Northern blot ( image 3) can determine that the microRNA mimic can be effectively and continuously expressed in the target cell, so as to ensure the subsequent steps of the technical system. Subsequently, streptavidin magnetic beads were used to capture the mRNA of the microRNA target gene in the crude cytoplasmic extract of the target cells after transfection, and the cDNA libraries of the target genes of miR-7 and miR-122 were established. The PCR results showed that the microRNA The fragments captured by the mimics were widely distributed in length, but each had different ...

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Abstract

The invention relates to a novel method for screening microRNA (Ribose Nucleic Acid) target genes at a high flux, in particular to a novel method for screening microRNA target genes at a high flux by using a biotin-modified microRNA mimic. The method is mainly characterized in that: the biotin-modified microRNA mimic is designed and synthesized, and the target gene mRNA which is directly combined with the biotin-modified microRNA mimic is enriched and selected by using a magnetic bead which contains a gametophyte with high affinity to biotins. By adopting the method, the mRNA target genes which directly interact with microRNA are enriched, so that problem of false positive existing in other methods is solved; moreover, the method is easy and convenient to operate, has relatively low cost and is suitable for most laboratories.

Description

technical field [0001] The invention relates to a new method for high-throughput screening of microRNA target genes using biotin-modified microRNA mimics. Background technique [0002] MicroRNA (miRNA for short) is a kind of important RNA with a length of 21-23nt related to post-transcriptional gene silencing, which was first discovered in Caenorhabditis elegans[1]. It has been confirmed that miRNA widely exists in eukaryotic cells and is one of the largest gene families, accounting for about 1%-4% of the entire genome [2]. Recent studies have shown that miRNA has a wide range of functions and plays a key role in various biological processes and regulatory pathways in organisms, such as developmental process control, cell growth and apoptosis, cell division, cell differentiation and organ development, virus infection, Maintain the dynamic balance of the body's physiological activities [3]. Moreover, the dysregulation of some important miRNAs will lead to dramatic changes i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 刘德培李振亚习杨朱文楠曾超吕湘
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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