miRNA detection and imaging method, composition and kit

A detection method and kit technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of high noise, probe selectivity, low sensitivity, lack of reliable solutions, etc. The effect of expanding the scope of application

Active Publication Date: 2022-02-25
SUZHOU UNIV OF SCI & TECH
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH technology still has some problems: such as probe selectivity, low sensitivity, and high noise in imaging analysis; more importantly, FISH detection usually requires cell immobilization, and can only detect dead cells at a specific time point microRNA expression level
In addition to the complex probe design required for nucleic acid nanoprobes, the fluorescent labeling of the probes also increases the cost; the uniformity and potential toxicity of noble metal nanoprobes limit their applications to some extent.
In summary, the development of a new in situ imaging method with high sensitivity and specificity, and little or no interference to the physiological process of the cell itself, can realize the monitoring of the spatial distribution and expression level of miRNAs, and explore the spatial distribution and expression of cellular gene expression. Heterogeneity has always been the goal pursued by researchers, but now there is a lack of reliable solutions

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
  • miRNA detection and imaging method, composition and kit
  • miRNA detection and imaging method, composition and kit
  • miRNA detection and imaging method, composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The present invention of this embodiment provides a miRNA detection method, the method is: in the intracellular environment, when the target miRNA to be detected exists, the target miRNA induces an entropy-driven circular reaction between the substrate probe and the dye probe , to generate the G-4 chain structure of the RNA aptamer Corn, so that the DFHO, which is originally non-fluorescent in the system, produces fluorescence, so that the detection of the target miRNA can be realized through the fluorescence intensity of the detection system.

[0052] This embodiment also provides a miRNA imaging method, the method is: in the intracellular environment, when the target miRNA exists, the target miRNA induces an entropy-driven circular reaction between the substrate probe and the dye probe to generate an RNA adapter The G-4 chain structure of the body Corn makes the DFHO, which is originally non-fluorescent in the system, produce fluorescence, so that the imaging of the ta...

Embodiment 2

[0054] The present invention also provides a composition for detecting and imaging miRNA, which is used for detecting and / or imaging target miRNA by using the method in Example 1.

[0055] In this embodiment, the target miRNA is miRNA-141, and its sequence is:

[0056] SEQ ID Nos 1: 5'-UAACACUGUCUGGUAAAGAUGG-3';

[0057] The composition includes:

[0058] Primer probe OB, its sequence is:

[0059] SEQ ID Nos 2: 5'-CGAAGAAGGA GGTCTGAGGAGGTCACTG-3';

[0060] Primer probe SB, its sequence is:

[0061] SEQ ID Nos 3:

[0062] 5'-CCACATACATCATATT AACT TAACACTGTCTGGTAA-3';

[0063] Primer probe LB, its sequence is:

[0064] SEQ ID Nos. 4:

[0065] 5'-CCATCTTTACCAGACAGTGTTAAGTTCAGTGACCTCCTCAGACC-3';

[0066] and dye probe F, whose sequence is:

[0067] SEQ ID Nos 5:

[0068] 5'-GGTCTGAGGAGGTCACTGAACTTAACACTGTCTGGTAA-3';

[0069] Among them, the primer probes OB, SB, and LB are used to hybridize and synthesize the substrate probe S, and the substrate probe S and dye probe F ...

Embodiment 3

[0076] The present invention also provides a kit for miRNA detection and imaging, which comprises the composition as described above.

[0077] In a preferred embodiment, the kit also includes TE buffer, RNase-free centrifuge tube, MgCl 2 solution, opti-MEM medium, lipo3000 solution.

[0078] In a preferred embodiment, the kit is used for target miRNA detection or imaging methods comprising the following steps (target miRNA and each probe sequence as shown in Example 2):

[0079] 1) Synthetic substrate probe S:

[0080] TE buffer (Tris-HCl 10 mM, EDTA 1 mM) was used to prepare primer-probe OB solution, primer-probe SB solution and primer-probe LB solution with a concentration of 100 μM, and then pipette 80 μL of each of the three primer-probe solutions into the In a 1.5ml RNase-free centrifuge tube, mix well and then add 2.4μL 1M MgCl 2 Solution, heated to 95°C and kept for 5 minutes, then cooled to room temperature, finally purified with a DNA purification kit, and dried to...

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 discloses a miRNA detection and imaging method, a composition and a kit, in an intracellular environment, when target miRNA exists, the target miRNA induces a substrate probe and a dye probe to generate an entropy-driven circular reaction to generate a G-4 chain body structure of an RNA aptamer Corn, so that DFHO which does not have fluorescence originally in a system generates fluorescence, therefore, the detection of the target miRNA is realized through the fluorescence intensity of the detection system, or the imaging of the target miRNA is realized through the fluorescence imaging system. A new scheme for high-sensitivity monitoring of miRNA in a biological system is constructed by utilizing a principle that a target miRNA induces a substrate probe and a dye probe to generate an entropy-driven circular reaction in an intracellular environment to generate a space structure of G-quadruplex Corn, so that DFHO which originally does not have fluorescence in the system generates fluorescence. And in-situ imaging and high-sensitivity and high-specificity detection of miRNA can be realized.

Description

technical field [0001] The invention relates to the field of nucleic acid sequence detection, in particular to a miRNA detection and imaging method, composition and kit. Background technique [0002] MicroRNAs (microRNAs, miRNAs) are a class of endogenous non-coding single-stranded RNAs widely present in animals and plants, consisting of 19-23 nucleotides. It can inhibit post-transcriptional gene expression by specifically binding to target messenger ribonucleic acid, and plays an important role in regulating gene expression, cell cycle, and organism development timing. It is an intracellular marker with extremely important pathological and physiological significance. Cell heterogeneity leads to differences in the growth rate, invasion ability, and drug sensitivity of each cell. To understand the heterogeneity information of some key genes in the cell tissue and to study its production process, it is not only necessary to understand its related microRNA and other markers In...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841C12N15/11
CPCC12Q1/6841C12Q2525/207C12Q2563/107C12Q2565/601Y02A50/30
Inventor 谷雨高唐杰龚梅影王小宝郭春显李长明
Owner SUZHOU UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap