Identification method of competitive endogenous RNA (Ribonucleic Acid) network

An identification method and competitive technology, applied in the field of identification of competitive endogenous RNA networks, can solve the problem of inability to verify whether different transcripts constitute a competitive regulatory relationship, inability to more accurately clarify the regulatory mechanism of different transcripts, and ceRNA network regulation Issues that have not yet been clearly understood, to achieve the effect of good sequence splicing, fast sequencing speed, and high sensitivity

Inactive Publication Date: 2017-08-25
成都生命基线科技有限公司 +1
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, various RNA and small RNA sequencing methods are very mature. Various transcripts with significant expression differences in various samples can be found through sequencing methods, but the relationship between them cannot be identified, and different transcripts cannot be elucidated more accurately. Regulatory Mechanism Between Books
Many software and methods such as miRanda and TargetScan have been used to find microRNA response elements in different transcripts, but they cannot verify whether different transcripts with the same microRNA response element constitute a competitive regulatory relationship
Not only that, the relationship between the ceRNA regulatory network and actual biological problems, for example, the specific mechanism through which the ceRNA network plays a regulatory role, and how the ceRNA network is regulated in vivo has not yet been clearly understood

Method used

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  • Identification method of competitive endogenous RNA (Ribonucleic Acid) network
  • Identification method of competitive endogenous RNA (Ribonucleic Acid) network
  • Identification method of competitive endogenous RNA (Ribonucleic Acid) network

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Build a sequencing library

[0046] Human liver cancer cells (HepG2) were resuscitated, saturated with DMEM medium (Gibco) containing 10% fetal bovine serum (Hyclone), 100U / mL penicillin and 100U / mL streptomycin (Hyclone) at 37°C, 5% CO2 Cultured in a humidified cell culture incubator. Cells in the logarithmic phase were inoculated in 60mm culture dishes at a density of 5.0×106 cells / mL, and when they grew to about 80%, HepG2 cells were treated with 1 μg / mL doxorubicin (Beijing Huafeng) in the experimental group, and HepG2 cells in the control group were added Equal volume of PBS. After 24 hours, cells were collected by centrifugation after 0.25% (mass percent) trypsinization. The collected HepG2 cells were extracted according to the experimental instructions of TRIzol (Invitrogen), and the total RNA of the cells was extracted, and then the purity and integrity of the RNA were identified. Then send samples for sequencing. After the sample is qualified, the library is ...

Embodiment 2

[0050] Screen differentially expressed mRNAs and lncRNAs as well as microRNAs

[0051] The original images obtained by HiSeq 4000 are converted into original sequencing sequences through base calling analysis, and the results are stored in the FASTQ file format. Filter reads with adapters and low quality in the original sequencing sequence to obtain CleanReads. Use Hisat2 (2.03-beta) to align clean reads to the human reference genome (hg19), use Cufflins (1.3.0) to assemble transcripts, use cuffmerge to fuse multiple annotation files generated, and use Cuffdiff to identify differentially expressed RNAs, Calculated by edgeR, according to q value <0.05, |log2(Fold change)|≥1 as the threshold value to screen to obtain differentially expressed lncRNA and mRNA respectively.

[0052] From the GEO (Gene Expression Omnibus) database, the sequencing data of p53-regulated microRNA in liver cancer cell HepG2 were obtained by small RNA sequencing (Small RNA-seq). v1.5) Statistical reads...

Embodiment 3

[0055] Verification of the accuracy of sequencing results

[0056] HepG2 cells were cultured to about 80% in a 10cm culture dish according to the culture conditions in Example 1. After 0.25% (mass percentage) trypsinization, the 5 cells / well were plated in 60 mm Petri dishes and cultured overnight. When the cells grew to about 70%, the control group was added with DMEM medium containing 10% FBS, and the experimental group was added with doxorubicin at a final concentration of 1 μg / mL, and cultured in a 37°C, 5% CO2 incubator. After 48 hours, 0.25% (mass percent) trypsin was digested and centrifuged to collect the cells, and the total RNA was extracted with Trizol, and the RNA was reverse-transcribed into cDNA using oligo(dT) primers and M-MLV reverse transcriptase, and some mRNAs and lncRNAs were randomly detected expression level to verify the accuracy of the sequencing results.

[0057] By doxorubicin treatment, the activity of p53 in the cells was increased. The qRT-PCR ...

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Abstract

The invention discloses an identification method of a competitive endogenous RNA (Ribonucleic Acid) network, and relates to the technical field of biology. The identification method for the competitive endogenous RNA network comprises the following steps: extracting total RNA from a cell, identifying the total RNA, constructing a library, and sequencing; screening differential expression transcripts; screening differential expression microRNA; respectively identifying the microRNA binding site of each differential expression transcript; detecting the competition relationship of each differential expression transcript on the binding site so as to identify a gene expression competitive endogenous RNA network. According to the method, classical regulatory elements, including transcription factors and the like, are combined with a competitive endogenous RNA hypothesis, a complex regulatory process which occurs in the cell is searched from a new perspective, and the mechanism of action of different regulatory elements in the cell is better illuminated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying a competitive endogenous RNA network. Background technique [0002] Competitive endogenous RNA (ceRNA) hypothesis is a brand-new hypothesis of gene expression regulation model proposed in recent years. Its core content is: MicroRNA (miRNA) is a kind of endogenous small molecule single-stranded RNA length About 20-24 nucleotides, relatively conserved in biological evolution, does not encode protein. Transcripts such as mRNA, pseudogene transcripts and long non-coding RNA (lncRNA) compete to bind the same microRNA through microRNA response elements to regulate their expression levels, thereby affecting the function of cells. [0003] At present, various RNA and small RNA sequencing methods are very mature. Various transcripts with significant expression differences in various samples can be found through sequencing methods, but the relationship between them ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G06F19/18
CPCC12Q1/6869G16B20/00C12Q2525/207C12Q2535/122
Inventor 郭志云白丽军熊莉丽凌之浩张一鸣庄振华
Owner 成都生命基线科技有限公司
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