Method for efficiently purifying and identifying cyclic RNA based on second-generation Illumina sequencing platform

A sequencing platform and circular technology, applied in the field of molecular biology to improve removal efficiency

Inactive Publication Date: 2019-09-27
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Circular RNA library construction generally eliminates linear RNA by adding RNase R, but

Method used

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  • Method for efficiently purifying and identifying cyclic RNA based on second-generation Illumina sequencing platform
  • Method for efficiently purifying and identifying cyclic RNA based on second-generation Illumina sequencing platform
  • Method for efficiently purifying and identifying cyclic RNA based on second-generation Illumina sequencing platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Extraction of total RNA

[0037] Take 100-150 mg of the ground sample, use the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen, Cat.#DP441) to extract the total RNA of the sample, and then use the DNA digestion enzyme gDNA Eraser to remove the genomic DNA. box instructions. Detect RNA bands by 1% agarose gel electrophoresis. If the brightness of 28S rRNA is 1.5-2 times that of 18S rRNA, it means that the RNA is not degraded. RNA concentration and absorbance were measured using a NanoDrop 2000c UV-Vis spectrophotometer, and the A260 / 280 was between 1.8-2.0, indicating that the purity of the RNA sample was high. The Agilent 2100 bioanalyzer was used to further detect the integrity number (RNA integrity number, RIN) of the RNA sample. If the RIN value was greater than 7.5, the sequencing requirements were met. Take 10ug of good quality total RNA for library construction experiments.

Embodiment 2

[0038] Example 2 Removal of polyadenylated mRNA in total RNA

[0039] (1) Oligo-dT Dyna magnetic beads pretreatment

[0040] 1) Use Dynabeads mRNA Purification kit (Invitrogen, Cat.61006) to thoroughly resuspend Oligo-dT Dyna magnetic beads, evenly pipette 200 μL into a new RNase-free tube, and place the tube on the magnetic stand for 1 -2min, the magnetic beads will gather at the magnetic end, transfer the supernatant with a pipette, discard it, and keep the tube on the magnetic stand at the same time.

[0041] 2) Resuspend Oligo-dT Dyna magnetic beads with 200 μL Binding Buffer, remove the tube from the magnetic stand, gently pipette several times to mix, put it back on the magnetic stand, let it stand for 1-2min, carefully transfer the supernatant, and discard . Repeat this step twice.

[0042] 3) Resuspend the Oligo-dT Dyna magnetic beads in 200 μL Binding Buffer again, and divide them equally into RNase-free tube 1 and tube 2, 100 μL in each tube.

[0043] (2) Removal...

Embodiment 3

[0059] Example 3 Ribosomal RNA removal

[0060] (1) Ribosomal RNA and probe hybridization

[0061] 1) Set the temperature of two water baths to 70°C and 37°C respectively

[0062] 2) Add to 1.5mL RNase-free tube

[0063]

[0064] The ribosome probe and Hybridization buffer are provided in the RiboMinus™ Plant Kit for RNA-Seq (Invitrogen, A1083808).

[0065] 3) Water bath at 70°C for 5 minutes to denature the RNA.

[0066] 4) Transfer the sample to a 37°C water bath for 30 minutes to slowly cool the RNA down to 37°C

[0067] 5) During the cooling process, pretreat the magnetic beads.

[0068] (2) Magnetic bead pretreatment

[0069] 1) Vortex the magnetic beads in the vial, pipette 750μL into a 1.5mL tube, place on the magnetic stand for 1min, remove the supernatant

[0070] 2) Add 750 μL DEPC water to wash, slowly vortex to resuspend, place on the magnetic stand for 1min, remove the supernatant, repeat this step

[0071]3) Add 750μL Hybridization buffer and slowly vor...

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Abstract

The invention discloses a method for efficiently purifying and identifying cyclic RNA based on a second-generation Illumina sequencing platform and belongs to the field of molecular biology. By use of a cyclic RNA sequencing library obtained by purification through the method, the removing efficiency of unrelated linear RNA can be greatly improved, and high-purity cyclic RNA is obtained by enriching. A virtual data set of the cyclic RNA is constructed on the basis of genome annotation. By searching and comparing reads of sequencing data, the types of the identified cyclic RNA are far higher than that of a conventional method. The method has important significance for researching an internal structure of the cyclic RNA and revealing the functions of the cyclic RNA.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for efficiently purifying and identifying circular RNA based on a second-generation Illumina sequencing platform. Background technique [0002] Circular RNA (circular RNA) is a type of closed circular RNA molecule that commonly exists in organisms. Unlike linear RNA, the 3' end and 5' end of circular RNA are closed. The expression level of circRNA is usually relatively low, and it shows obvious cell type specificity and tissue specificity. Their biosynthesis depends on the splicing machinery and is regulated by cis-complementary sequences and protein factors. Studies have shown that some circRNAs are closely related to neuronal function, innate immune response, cell proliferation and cell pluripotency. At the molecular level, they affect gene expression by binding to microRNAs or proteins, regulating RNA polymerase II (Pol II) transcription, and interacting...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2525/173C12Q2525/307C12Q2535/122C12Q2537/165
Inventor 王慧慧王永生王汇源席飞虎韩茜妹顾连峰
Owner FUJIAN AGRI & FORESTRY UNIV
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