A kind of circular RNA forming sequence and application

A DNA sequence and sequence technology, applied in the field of DNA sequence to help circular RNA form a circle, can solve the problems that circRNA has not been discovered and has not been studied, and achieve the effect of stable results and easy operation

Active Publication Date: 2019-09-10
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the emerging of this field, most circRNAs have not been discovered or studied

Method used

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  • A kind of circular RNA forming sequence and application
  • A kind of circular RNA forming sequence and application
  • A kind of circular RNA forming sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, the construction of the plasmid of pcCirc

[0063] The technologies involved in the present invention are conventional technical means of molecular cloning, and the enzymes, primers, reagents and reaction conditions involved in them can be reasonably selected according to the experience of those skilled in the art unless otherwise specified, and the reagent consumables involved belong to Commercially available common products, detection means and instruments involved therein are also well known and mastered by those skilled in the art. The technical solution of the present invention will be further described through examples and test examples, but it should not be construed as a limitation of the present invention.

[0064] The circular RNA looping sequence of the present invention includes a 5' looping sequence and a 3' looping sequence, and a multiple cloning site region between the two looping sequences, the 5' looping sequence consists of 45 bases, Th...

Embodiment 2

[0092] Example 2, overexpression of circRNF13 in tongue squamous cell carcinoma cells

[0093] 1. Materials and methods

[0094] 1.1 Cell culture and transfection

[0095] The tongue cancer cells Tca8113 and Cal27 in good growth state were divided into 2×10 5 Cells / well were seeded in a 6-well plate, and the 6-well plate was placed at 37°C, 5% CO 2 In the incubator, the transfection of the circRNF13 overexpression vector can begin when the cells to be cultured grow to a density of 50-70%; the transfection process is as follows:

[0096] Add 100 μl of the prepared polylysine-modified silicon nanoparticle suspension carrying the circRNF13 eukaryotic expression plasmid to a sterile EP tube, and gently mix with 100 μl of serum-free medium; wash the cells 3 times with D-Hank's solution; Add 800 μl of serum-free medium (without antibiotics) to the above mixture, mix gently and add to one well of a 6-well plate; place the 6-well plate in CO 2 Incubate at 37°C for 6 hours in an in...

Embodiment 3

[0109] Cell Culture and Transfection

[0110] Tongue squamous cell carcinoma cells Tca8113 and Cal27 in good growth state or drug-resistant cells were divided into 2×10 5 Cells / well were seeded in a 6-well plate, and the 6-well plate was placed at 37°C, 5% CO 2 In the incubator, the transfection of circRNF13 overexpression vector or siRNA can be started when the cultured cells grow to 50-70% density; the transfection process is as follows:

[0111] Add 100 μl of prepared polylysine-modified silicon nanoparticle suspension carrying circRNF13 eukaryotic expression plasmid or siRNA to a sterile EP tube, mix gently with 100 μl serum-free medium; wash cells with D-Hank's solution 3 times; add 800 μl serum-free medium (without antibiotics) to the above mixture, mix gently and add to 1 well of the 6-well plate; place the 6-well plate in CO 2 Incubate at 37°C for 6 hours in an incubator, discard the supernatant, and add complete medium to continue culturing overnight. Silicon nanop...

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Abstract

The invention discloses a circRNA ring-forming sequence and application. The sequence induces the non-circular structure RNA ring-forming expression through building expression vectors. The circRNA ring forming sequence is generally applicable to the ring forming expression of various circRNA; the expression efficiency is high and stable; a plurality of restriction enzyme cutting sites exist inside the sequence; the most circRNA expression requirement can be met; in addition, the sequence can be integrated to various kinds of expression vectors and can be applied to various expression systemssuch as ordinary eukaryotic expression, lentiviral expression, adenovirus expression, retrovirus expression and prokaryotic expression; when the vectors containing the sequence are used for expressingcircRNA, the operation is simple; the implementation is easy; the popularization is easy. The invention provides a powerful study tool for studying the function and mechanism of the circRNA; the theoretical support is provided for determining the study and development of the circRNA molecules as novel markers and disease treatment targets.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA sequence for helping circular RNA form a circle and its application. Background technique [0002] Human Genome Project and its follow-up DNA Elements Encyclopedia Project (The Encyclopedia of DNAElements Project, ENCODE) research results show that protein-coding gene sequences only account for 1-3% of the human genome sequence, while most of the human genome can be transcribed The sequence is non-coding RNA (non-coding RNA, ncRNA). Although non-coding RNA does not encode protein, it has been attracting much attention in the field of biomedical research due to its extensive involvement in the regulation of gene expression in cells. The most studied are linear ncRNA molecules, which can be divided into microRNA (microRNA, miRNA, 19-23nt) and long non-coding RNA (long non-coding RNA, lncRNAs, >200nt) according to the length of the RNA sequence. It plays an important role in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66
Inventor 郭灿熊芳曾朝阳熊炜刘凌云李勇王裕民莫勇真廖前进周钰娟李小玲李桂源
Owner CENT SOUTH UNIV
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