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Method for detecting differential expression of circRNA equipotential sites of plant in high throughput manner

A high-throughput, plant-based technology, applied in biochemical equipment and methods, DNA/RNA fragments, DNA preparation, etc., can solve the problem of high-throughput and accurate analysis of circRNA allelic expression.

Active Publication Date: 2019-02-22
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the commonly used allelic imbalance expression detection mainly focuses on protein-coding genes, but there is no high-throughput and accurate analysis method for the allelic expression of circRNAs that widely exist in transcriptome data.

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  • Method for detecting differential expression of circRNA equipotential sites of plant in high throughput manner
  • Method for detecting differential expression of circRNA equipotential sites of plant in high throughput manner
  • Method for detecting differential expression of circRNA equipotential sites of plant in high throughput manner

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Experimental program
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Effect test

Embodiment 1

[0052] Take the fresh leaves of Populus tomentosa, use the RNA extraction kit (MagJ ET Plant RNA Purification Kit, No.K2772) to carry out total RNA extraction, use Ribo-Zero TM rRNA Removal Kits (Plant) kit (No. MRZPL116) remove rRNA, obtain Poly(A)-RNA samples, use RNase Rd to digest linear RNA (reaction system: RNA, 5μg; 10X ReactionBuffer, 5μL; RNase R, 20U ; RNase-Free water, supplemented to 50 μ L), obtain Poly(A)- / Ribo-RNA sample, utilize SMART kit (SMART cDNALibrary ConstructionKit, NO.634901) to carry out strand-specific cDNA library construction;

[0053] Using Illumina HiSeq TM 2500 for double-end sequencing, the sequencing data volume is 12G. Adapters and redundant sequences were removed, and transcripts were spliced ​​with the default parameters of cufflinks software. Use find_circ to compare the sequences that have not been compared to the reference sequence (the reference sequence is the genome sequence of Populus trichocarpa V3.0 https: / / phytozome.jgi.doe.gov...

Embodiment 2

[0070] The leaves of Populus microphylla treated with high temperature were used for total RNA extraction, using RNA extraction kit (MagJ ET PlantRNAPurification Kit, No.K2772), using Ribo-Zero TM The rRNA Removal Kits (Plant) kit (No.MRZPL116) removes rRNA, and then uses the magnetic bead method to bind Poly(A) RNA to obtain Poly(A)-RNA samples, and uses RNase Rd to digest linear RNA, (reaction system : RNA, 5 μg; 10X Reaction Buffer, 5 μL; RNase R, 20U; RNase-Free water, supplemented to 50 μL), to obtain Poly(A)- / Ribo-RNA samples, using SMART kit (SMART cDNA Library Construction Kit, NO .634901) for strand-specific cDNA library construction;

[0071] Using Illumina HiSeq TM2500 for double-end sequencing, the sequencing data volume is 12G. Adapters and redundant sequences were removed, and transcripts were spliced ​​by cufflinks software. Use find_circ to extract 20-nt anchor sequences at both ends of the reads that are not aligned to the reference sequence, and align eac...

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Abstract

The invention provides a method for detecting differential expression of circRNA equipotential sites of a plant in a high throughput manner, and belongs to the technical field of detection for gene expression. The method comprises the following steps: 1) extracting total RNA of a plant sample and constructing a strand specificity library; 2) performing paired-end sequencing on the strand specificity library with Illumina HiSeq; 3) screening circRNAs data from original sequencing data; 4) extracting reverse splicing reads at a circRNAs looping position from the circRNAs data; 5) detecting mononucleotide variation of the reverse splicing reads; and 6) counting the number of the compared reads with different genotypes at SNP (single nucleotide polymorphism) spots in the reverse splicing readsand taking a proportion of the number of the compared reads with different genotypes as a proportion of expression quantities of different genotypes. According to the method, the differential expression of the circRNA equipotential sites can be accurately detected in a high throughput manner.

Description

technical field [0001] The invention belongs to the technical field of gene expression detection, in particular to a method for high-throughput detection of differential expression of plant circRNA allelic sites. Background technique [0002] Allele (also called allelomorph) generally refers to a pair of genes that control relative traits at the same position on a pair of homologous chromosomes. [0003] The unbalanced expression of alleles (allelic expression imbalance, AEI) is that in the same cell, each gene usually has 2 copies, and the expression ratio of the 2 copies of the gene deviates from 1:1 due to cis-action. The phenomenon of unbalanced expression of alleles is ubiquitous. In addition to the absolute unbalanced expression of genetically imprinted genes, a considerable number of genes have AEI in some individuals and in different time and space in the same individual. And associated with polymorphic sites in some specific regions of the genome. [0004] At pres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/113
CPCC12Q1/6895C12Q2600/156C12Q2600/178G16B20/20G16B25/10G16B30/10C12N15/1072
Inventor 张德强宋跃朋轩安然卜琛皞
Owner BEIJING FORESTRY UNIVERSITY