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A method for detection of miRNAs using Digoxigenin-labeled EDC cross-linking and bridging

A digoxin labeling and cross-linking bridge technology, applied in the field of detection of miRNAs, can solve the problems of low sensitivity, high price, complicated procedures, etc., and achieve the effect of low detection cost

Inactive Publication Date: 2014-10-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to disclose a method for detecting miRNAs using digoxin-labeled EDC cross-linking and bridging method, which solves the problems of low safety, complicated procedure, high price and low sensitivity of the current detection method

Method used

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  • A method for detection of miRNAs using Digoxigenin-labeled EDC cross-linking and bridging
  • A method for detection of miRNAs using Digoxigenin-labeled EDC cross-linking and bridging
  • A method for detection of miRNAs using Digoxigenin-labeled EDC cross-linking and bridging

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Embodiment 1

[0042] Detection of hsa-miR-21 in HeLa cells by DSLE

[0043] In order to verify the feasibility of the DSLE method, the author chose hsa-miR-21 highly expressed in Hela cells as the research object, its sequence is 5'-phos-uagcuuaucagacugauguuga-3', and the corresponding hsa-miR-21 Bridge sequence is

[0044] 5'-GAATGTCATAAGCGTCAACATCAGTCTGATAAGCTA-3'; 3'DIG-probe

[0045] 5'-phos-CGCTTATGACATTC-DIG-3', all synthesized by Shanghai Bioengineering Co., Ltd.

[0046] 1) Extraction of total RNA from HeLa cells

[0047] The total RNA of HeLa cells was extracted with Trizol according to the instructions, and the measured concentration was 1 μg / μl.

[0048] 2) Capture and connection of hsa-miR-21

[0049] Take 2μg and 1μg total RNA for the miRNAs capture experiment, and set up a negative control group without RNA or Bridge. The capture system is as follows: 2μl HeLa RNA+5μl ddH 2 O (Rnase free) or 1μl HeLa RNA+6μl ddH 2 O (Rnase free), 1μl 1pmol / μl hsa-miR-21 Bridge, 1μl 10×Cap...

Embodiment 2

[0072] DSLE method to detect low abundance Giardia miRNA

[0073] Giardia miR2 is produced by Giardia snoRNA17 (GlsR17). Studies have shown that its expression level in Giardia trophozoites is very low, and the traditional radioactive Northern blots method cannot detect this miRNA. In order to verify whether the DSLE method can detect low-abundance expressed miRNAs, Giardia miR2 was used as the research object in this experiment, and the DSLE method was used for detection. The Giardia miR2 sequence is 5'-CAGCCUAAUCACCGCCCCUAUAGUCC-3', and the corresponding Giardia miR2 Bridge sequence is

[0074] 5'-GAATGTCATAAGCGGGACTATAGGGGCGGTGATTAGGCTG-3'; 3'DIG-probe

[0075] 5'-phos-CGCTTATGACATTC-DIG-3', all synthesized by Shanghai Bioengineering Co., Ltd.

[0076] 1) Extraction of total RNA from Giardia cells

[0077] The total RNA of Giardia lamblia trophozoites was extracted with Trizol according to the instructions, and the measured concentration was 1 μg / μl.

[0078] 2) Cap...

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Abstract

The invention discloses a method for detecting miRNAs by a DIG labeling EDC (carbodiimide) cross-linking bridging method. Once a DIG labeling system is utilized, the sensitivity of the DIG labeling system is improved by at least 50 times in contrast with that of a traditional DIG labeling Northern blots detection system; and the sensitivity of the DIG labeling system is equivalent with that of a radioisotope labeling Northern blots detection system; the method cannot cause any pollution on environment, and is very safe and free of a hybridization step; all operations can be completed in 9-10 hours; and therefore, the method is convenient, quick, and low in detection cost. As a result, the problems of low safety, complex procedure, high price, low sensitivity and the like of a current detection method are solved.

Description

technical field [0001] The invention provides a method for detecting miRNAs by using digoxin-labeled EDC cross-linking and bridging method, which can detect microRNAs in a non-radioactive, rapid and high-sensitivity manner. Background technique [0002] In recent years, one of the most important discoveries in biomedical research is the discovery of non-coding microRNAs (miRNAs) with a size of about 22 nt. These small RNA molecules are not translated into proteins, but can act on mRNA through complementary base pairing, degrading target mRNA or inhibiting translation. It is estimated that only about 1% of the genome of mammalian cells encodes miRNAs molecules, and these miRNAs regulate 1 / 3 of the coding genes of mammals, which shows their importance to the life activities of cells. [0003] Studying the temporal and spatial expression profiles of miRNAs in organisms can enable researchers to better understand the biological functions of miRNAs. To achieve this goal, many t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 张西臣李建华吴威宫鹏涛杨举张楠李赫杨正涛张国才
Owner JILIN UNIV
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