Supercharge Your Innovation With Domain-Expert AI Agents!

Method and kit for detecting m<7>G (N7-methylguanosine) modification in RNA with single base resolution in whole transcriptome range

A transcriptome, single-base technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as increased harm and inapplicability

Active Publication Date: 2018-11-20
INST OF ZOOLOGY CHINESE ACAD OF SCI
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, these two chemical reagent-based detection m 7 The method of G nucleoside site is detected by radioactive element labeling, which makes the method more harmful, and can only be applied to the detection of high-abundance tRNA and rRNA, and is not suitable for low-abundance mRNA and small RNA

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method and kit for detecting m&lt;7&gt;G (N7-methylguanosine) modification in RNA with single base resolution in whole transcriptome range
  • Method and kit for detecting m&lt;7&gt;G (N7-methylguanosine) modification in RNA with single base resolution in whole transcriptome range
  • Method and kit for detecting m&lt;7&gt;G (N7-methylguanosine) modification in RNA with single base resolution in whole transcriptome range

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Detection of RNA7mG modification in E. coli

[0032] 1. RNA acquisition

[0033] (1) Centrifuge the 100 mL of Escherichia coli cultured overnight, at 4000 rpm, 4°C for 5 minutes, remove the supernatant, and retain the bacterial pellet;

[0034] (2) Put the prepared mortar and pestle on a tray filled with water, pour a little alcohol and ignite it, take out the mortar and pestle after the alcohol is burned and cooled;

[0035] (3) Introduce liquid nitrogen into the mortar, use a syringe to suck the bacterial pellet and drop the precipitate into the liquid nitrogen drop by drop. After the liquid nitrogen is converted, use a pestle to grind and grind into powder, and then add liquid nitrogen. After the liquid nitrogen disappears, immediately use a pestle to grind, repeat this three times, and finally add Trizol Reagent immediately (add more points), and then Trizol Reagent will condense into a block;

[0036] (4) After Trizol Reagent becomes liquid, pipette and mix well, ...

Embodiment 2

[0102] Example 2 Detection of RNA7mG modification in HEK293T cell line

[0103] 1. RNA acquisition

[0104] (1) Digest the cells cultured in a 10cm diameter cell culture dish with trypsin, collect them in a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, remove the supernatant, and save the cell pellet;

[0105] (2) Resuspend the cells in 10 mL PBS buffer, centrifuge for 5 minutes, remove the supernatant, and save the cell pellet;

[0106] (3) Add 2 mL of LTrizol Reagent to the cell pellet, pipette to mix, and then divide 1 mL per tube into 1.5 mL EP tubes, then shake and mix with a shaker, and place on ice for 5 minutes to make the cells full Cracking

[0107] (4) Add 1 / 5 chloroform according to the volume of TRI Reagent, mix vigorously with a shaker for 15 seconds, and place on ice for 10 minutes;

[0108] (5) Centrifuge at 12,000rpm for 15 minutes at 4°C, transfer the upper aqueous phase to a new EP tube, taking care not to absorb the middle organic solvent layer. Add an ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method and a kit for detecting m<7>G (N7-methylguanosine) modification in RNA with single base resolution in a whole transcriptome range, and belongs to the technical field ofgene sequencing. The method comprises steps as follows: whole transcriptome RNA of sample cells is broken into fragmented RNA; after fragmented RNA is reduced by sodium borohydride, reduced RNA is broken by aniline hydrochloride, and sequencing adapters are connected with the two ends of broken RNA; after reverse transcription, PCR amplification is performed by sequencing primers to construct a sequencing library; the sequencing library is subjected to sequencing, and sequencing data is obtained; the sequencing data is analyzed, a ratio of RNA breakpoint number to sequencing depth is obtainedand is larger than 0.5, and the breakpoint position which is a G base site is determined as the m<7>G site. According to sequencing result analysis, not only can distribution of the m<7>G modification in transcriptome be analyzed, but also the specific site of the m<7>G modification in the transcriptome can be determined.

Description

Technical field [0001] The invention belongs to the technical field of gene sequencing, and in particular relates to a single-base resolution detection of RNA internal m 7 G modification method and kit. Background technique [0002] As a link between DNA and protein in the central law, RNA is not only responsible for transmitting genetic information, but also has a variety of post-transcriptional regulatory functions, but they are often overlooked in previous studies. In recent years, with the continuous advancement of research methods, including the innovation of sequencing technology, mass spectrometry and other analytical methods, as well as the introduction of chemical biology research ideas, people have already discovered a lot of RNA modifications that have not attracted enough attention. Research interest. [0003] Initially, when people analyze various modifications in RNA, they can only perform qualitative and quantitative analysis through instrumental analysis methods su...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2535/122
Inventor 藤花景陈云梁加龙孙中生
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com