Circular RNA-MET gene in liver cancer as well as expression method and fluorescent quantitative PCR detection method of circular RNA-MET gene

A technology of fluorescence quantitative detection and expression method, which is applied in the direction of DNA / RNA fragments, DNA preparation, recombinant DNA technology, etc., can solve the problems of no further research and achieve high specificity

Active Publication Date: 2015-11-04
GUANGZHOU GENESEED BIOTECH +1
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Problems solved by technology

Circularization of the RNA of the MET gene was found by searching the specialized circular RN

Method used

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  • Circular RNA-MET gene in liver cancer as well as expression method and fluorescent quantitative PCR detection method of circular RNA-MET gene
  • Circular RNA-MET gene in liver cancer as well as expression method and fluorescent quantitative PCR detection method of circular RNA-MET gene
  • Circular RNA-MET gene in liver cancer as well as expression method and fluorescent quantitative PCR detection method of circular RNA-MET gene

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Embodiment 1

[0100] A method for expressing circular RNA-MET gene in liver cancer, comprising the following steps

[0101] 1) extract the total RNA in cells and tissues, according to The Reagent reagent operating instructions extract the total RNA in liver cancer cells and liver cancer tissues; the detailed steps of extraction are:

[0102] a) Take a 2mg tissue piece from liver cancer tissue, grind the tissue with liquid nitrogen until pulverized, transfer it to a 1.5ml centrifuge tube, add 1ml trizol; take about 1 million cells from liver cancer cells, add 1ml trizol;

[0103] b) Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 15 minutes;

[0104] c) Centrifuge at 12000g for 15min at 4°C, the solution is divided into three layers, the RNA is dissolved in the water phase, and the water phase is transferred to another new RNase free EP tube;

[0105] d) Add 1 volume of isopropanol, vortex and mix thoroughly;

[0106] e) Centrifuge at 1200...

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Abstract

The invention discloses a circular RNA-MET gene in liver cancer as well as an expression method and a fluorescent quantitative PCR (polymerase chain reaction) detection method of the circular RNA-MET gene. The cDNA nucleotide sequence of the circular RNA-MET gene in the liver cancer is shown by SEQ ID NO:1. By designing a specific primer capable of amplifying the circular RNA, the circular RNA of the MET gene is amplified out by PCR, and an accurate cyclization site of circular RNA-MET is measured by a method for sequencing a PCR product namely cloned DNA; and the MET gene is determined to objectively contain the circular RNA consisting of 1214 nucleotides. By using the fluorescent quantitative PCR detection method of the circular RNA-MET gene in the liver cancer, the existence and expressive differences of the circular RNA-MET in samples of liver cancer cells and liver cancer clinical tissues can be specifically detected. The invention can provide an ideal gene detection method for early clinical diagnosis of liver cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a circular RNA-MET gene in liver cancer, an expression method thereof and a fluorescent quantitative PCR detection method; it provides a basis for early diagnosis and classification of liver cancer. Background technique [0002] Circular RNAs (circRNAs) in organisms are a class of RNAs with special and unknown functions, and exist in large numbers objectively. Circular RNAs are formed by shearing precursor RNAs and then head-to-tail connections of linear RNAs. Due to technical limitations in previous studies, this part of objectively existing RNAs has been ignored. With the development of deep RNA sequencing and large-scale biological With the development of information technology, researchers have really discovered that there are a large number of circularized RNA molecules in organisms. Due to the formation of closed loops, circularized RNAs are very stable in organisms. The speci...

Claims

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Application Information

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IPC IPC(8): C12N15/113C07K7/64C12N15/10C12Q1/68
Inventor 缪辉来张茂雷
Owner GUANGZHOU GENESEED BIOTECH
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