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rRNA capture probe and application thereof

A technology for capturing probes and probe sequences, applied in the field of rRNA capture probes, can solve one or more problems, achieve the effect of saving sequencing data volume and cost, and good application prospects

Pending Publication Date: 2020-03-13
SHENZHEN E GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above technical problems, the embodiments of the present invention provide an rRNA capture probe and its application to solve one or more problems existing in existing rRNA removal methods

Method used

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  • rRNA capture probe and application thereof
  • rRNA capture probe and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0091] Example 1: Prokaryotic transcription library sequencing analysis of cyanobacteria

[0092] 1.1) System6803 Pool Mix (the mixture of various rRNA capture probes shown in the gene sequence table) annealed with total RNA:

[0093] First, prepare Hybridization Buffer: 500mM Tris-HCl (pH 7.0), 1M NaCl. Take 200ng of cyanobacterial whole transcriptome RNA in a 0.2ml PCR tube, add Hybridization Buffer 3ul, Probe Mix 0.5ul, the total volume is 15ul. React on a PCR instrument at 95°C for 2 minutes, then gradually lower the temperature (0.1°C / sec) to 22°C, and react for 5 minutes. Immediately after the reaction, place it on an ice box to minimize RNA degradation.

[0094] 1.2) RNase H enzyme digestion:

[0095] In the PCR tube of the previous step reaction, Nuclease free water 1ul, RNase H Buffer 2ul, RNase H (5U / ul) 2ul, the total volume is 20ul. 37°C on the PCR instrument, the temperature of the hot cover is 47°C, and the reaction is 30min. Immediately after the reaction, ...

Embodiment 2

[0122] Example 2: Detection of trace RNA methylation based on probe removal of ribosomal rRNA:

[0123] 2.1) Preparation of magnetic bead-antibody complex:

[0124]Add 30ul protein A magnetic beads and 30ul protein G magnetic beads to a 1.5ml centrifuge tube, wash twice with 200ul immunoenrichment buffer, remove the supernatant; add 500ul to the centrifuge tube containing magnetic beads The immunoenrichment buffer, 5ug of anti-m6A antibody, 4 ℃ rotation reaction overnight.

[0125] 2.2) Antibody enrichment of m6A total RNA fragments:

[0126] Take 5ug of total RNA for fragmentation reaction in a 20ul system. The reaction condition is 70°C for 5min. After the reaction, 2ul of EDTA is added to terminate the reaction, and the fragmented RNA is recovered by ethanol precipitation with a length of 100-200bp.

[0127] Put the overnight reacted magnetic bead-antibody complex on a magnetic stand to remove the supernatant, wash the magnetic bead-antibody complex twice with 300ul of im...

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Abstract

The embodiment of the invention discloses an rRNA capture probe and an application method thereof. The rRNA capture probe is selected from one or more of the following three groups of probe sequences:a 5SrRNA probe sequence group including SEQ ID No. 1 and SEQ ID No. 2; a 16SrRNA probe sequence group including SEQ ID No. 3 to SEQ ID No. 24; and a 23SrRNA probe sequence group including SEQ ID No.25 to SEQ ID No. 76. Based on the capture probe, more than 90% of ribosomal RNA sequences in transcripts can be effectively removed; the technical problem that the prokaryotic mRNA does not contain polyA tail and cannot be enriched and separated by oligo dT is overcome; and the sequencing data volume and cost are greatly saved.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an rRNA capture probe and its application. Background technique [0002] Ribosomal RNA (rRNA) is the most abundant type of RNA in cells, accounting for more than 80% of all RNA in cells. It combines with proteins to form ribosomes, and under the guidance of the mRNA sequence, it transports specific amino acids to synthesize protein peptide chains. [0003] In prokaryotes, ribosomal RNA is mainly divided into three categories: 5S rRNA (about 120nt), 16S rRNA (about 1540nt) and 23S rRNA (about 2900nt). Eukaryotes are mainly divided into 5S rRNA (about 120nt), 5.8S rRNA rRNA (about 160nt), 18S rRNA (about 1900nt) and 28S (about 4700nt) rRNA. [0004] Due to the extremely high proportion of ribosomal RNA, ribosomal RNA will occupy a large amount of available data when studying the expression abundance and modification status of other functional RNAs in total RNA sequencing. ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6869
CPCC12Q1/6869C12Q2521/327C12Q2535/122C12Q2565/519
Inventor 王君文胡琪苏锦玲高飞
Owner SHENZHEN E GENE TECH
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