Novel miRNA detection technique and application thereof

A technology and a specific sequence technology, applied in the field of biomedicine, can solve the problems of reducing experimental sensitivity and specificity, technical complexity, and laboriousness, and achieve the effects of reducing experimental costs, high sensitivity, and low cost

Inactive Publication Date: 2010-08-11
江其生
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Problems solved by technology

[0007] 2.1 Northern blotting: Northern blotting is currently a common method for detecting miRNA expression. This method uses probes to hybridize with purified RNA to detect target molecules on solid supports (membranes). This technique is complex, laborious, and time-consuming
However, at present, most of the methods used in this method are based on the non-specific fluorescent labeling dye Sybrgreen, which reduces the sensitivity and specificity of the experiment.

Method used

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Examples

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Embodiment Construction

[0027] 1 miRNA extraction and purification

[0028] The miRNeasy Mini Kit (PN 217004) from QAIGEN Company was used to extract and separate large Total RNA (>200nt) and small RNA (<200nt), and the operation method was carried out according to the kit instructions. Determine the two RNA concentrations by measuring the 260nm OD value, and use 1% TAE agarose gel electrophoresis to confirm that the large RNA is not degraded to ensure the quality of the small RNA; you can also use 3-4% TAE agarose gel electrophoresis to confirm the small RNA.

[0029] 2 miRNA tailing

[0030] Take about 1-2 μg of miRNA, and use Poly(A) Tailing Kit (ABI, PN 1350M) to add A to the tail end, and the reagent dosage and operation method are carried out according to the kit instructions. The tailed small RNA is reverse transcribed into cDNA.

[0031] 3 miRNA reverse transcription

[0032] Reverse transcription was carried out with reverse transcriptase SuperScript III (Invitrogen, PN18080085) at 50°C f...

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Abstract

The invention relates to a preparation and technical method of a novel reagent (kit) required for detecting tiny RNA (miRNA) used in the field of biomedicine, and application thereof to the fields of clinical medicine, biology, molecular biology and the like. The invention belongs to the design, preparation and technical method application of the experiment reagent which uses specially designed primers and synthesized MGB probes for detecting and identifying mature miRNAs on the basis of a fluorescence quantitative PCR method. Compared with other methods, the technical method established by the invention has the characteristics of high sensitivity, high specificity, high throughput detection capability, short time consumption, relatively simple operation and the like. Thereby, the invention relates to the preparation of the novel miRNA detection reagent or reagent kit, the establishment of the technical method, the basic research of the reagent or the reagent kit, and the relevant prevention, treatment and research in clinical medicine such as malignant tumor, cardiovascular system diseases, nervous system diseases and the like.

Description

technical field [0001] The invention relates to the preparation, technical method and application of a new reagent (box) for detecting microRNA (miRNA) in the field of biomedicine and its application in the fields of clinical medicine, biology, molecular biology and the like. Specifically, it is a kind of experimental reagent design, preparation and technical method application based on fluorescent quantitative PCR technology for high sensitivity and high specificity detection and identification of mature miRNAs using MGB probes. Background technique [0002] 1 miRNA and its role [0003] Small RNAs are RNA regulatory molecules with a length of 19-28 nt, mainly including microRNA (microRNA, miRNA) and small interfering RNA (short-interfering RNA, siRNA). miRNAs are one of the new research hotspots after siRNAs, 2002 In 2003 and 2003, it was rated as one of the top ten scientific achievements of the year by the American "Science" magazine [Couzin J. et al. Science, 2002, 298...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 江其生
Owner 江其生
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