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In-situ combination detection method for miRNA expression and cell apoptosis

A combined detection and RNA probe technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as limited information and difficult detection, and achieve the effect of filling the application gap and good in-situ detection effect

Inactive Publication Date: 2008-08-13
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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Problems solved by technology

[0003] The average length of miRNA is only 22bp, which is difficult to detect by conventional methods
The miRNA real-time PCR and Northern blot developed in recent years have solved the detection of miRNA amplification; however, at the research level of miRNA subcellular localization and the mechanism of miRNA and target mRNA, the information that the above experimental methods can provide is limited. appear very limited

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  • In-situ combination detection method for miRNA expression and cell apoptosis

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preparation example Construction

[0014] 1. Preparation of LNA-miRNA probe

[0015] 1. Chemical synthesis of miRNA antisense RNA probes required for detection;

[0016] 2. Hybridize the probe synthesized above with an absolute excess of digoxin in a liquid phase environment overnight, and remove excess digoxin and RNA probes that do not participate in the reaction by means of chromatography and dialysis. The resulting solution was labeled as hybridization probe solution.

[0017] 2. Preparation of in situ hybridization reaction system

[0018] 1. Pepsin was made into a 10× stock solution for later use, and solution A was obtained;

[0019] 2. 40% deionized formamide, 10% dextran sulfate, 1×Denhardt solution, 4×SSC, 1g / L tRNA, 1g / L heat-denatured salmon sperm DNA, mixed uniformly to obtain solution B;

[0020] 3. Goat serum albumin was prepared into a solution with a concentration of 20% to obtain solution C;

[0021] 4. After activating biotin, combine with mouse anti-digoxigenin to obtain solution D;

[...

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Abstract

The invention discloses a method for in-situ joint detection of the expression of miRNA and fluorescence in-situ hybridization detection of apoptosis. The method comprises: synthesizing miRNA specific probe modified by LNA, labeling the probe with digoxin, purifying, biotinzing mouse anti-digoxin and constructing affinity-fluorescence labeling system and other basic steps. The invention can in-situ joint detect the expression of miRNA and apoptosis at the same time. The detection method of the invention converts the expression condition of miRNA to visible fluorescence signal, getting a good in-situ detection effect and filling a gap in the application of miRNA in-suit fluorescence detection.

Description

【Technical field】 [0001] The invention relates to the improvement of the miRNA in situ detection method. In particular, it is a fluorescent in situ hybridization detection method for in situ combined detection of miRNA expression and cell apoptosis. 【Background technique】 [0002] In recent years, studies have found that a new class of microRNA (microRNA or miRNA) refers to some special small non-coding RNAs with a length of about 21-25nt. By combining with its 3'untranslated region, it can negatively regulate gene expression by promoting the degradation of target mRNA and / or inhibiting the translation process at the post-transcriptional level. It is speculated that nearly 30% of human gene expression is precisely regulated by miRNAs. There are more than 300 miRNAs that have been identified so far. According to the speculation of the miRNA database Sanger, the total number may exceed 1000, accounting for 3% of the human genome. Because miRNAs exist widely in organisms and...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 康春生周旋浦佩玉
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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