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Method for detecting LRP6 to be target gene of miR-29a by adopting dual luciferase reporter gene

A dual-luciferase and reporter gene technology, applied in the field of detecting LRP6 as a target gene of miR-29a, can solve the problem of unproven signal pathway activation

Inactive Publication Date: 2018-03-30
THE UNIV OF HONG KONG SHENZHEN HOSPITAL
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Problems solved by technology

[0007] However, whether miR-29a can also activate the Wnt / -catenin signaling pathway by targeting its 3'UTR has not been confirmed

Method used

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  • Method for detecting LRP6 to be target gene of miR-29a by adopting dual luciferase reporter gene
  • Method for detecting LRP6 to be target gene of miR-29a by adopting dual luciferase reporter gene
  • Method for detecting LRP6 to be target gene of miR-29a by adopting dual luciferase reporter gene

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Experimental program
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Embodiment Construction

[0063] 1 Materials and methods

[0064] 1.1 Main reagents, instruments and consumables

[0065]

[0066]

[0067] 1.2 Solution preparation

[0068] 1.2.1 Preparation of Mglucose: Dissolve 36g of glucose (Glucose) in 90ul of deionized sterilized water, dilute to 100ml, and filter to sterilize with a 0.22um filter membrane.

[0069] 1.2.2 Preparation of liquid SOC medium: tryptone 8.0g, yeast extract 2.0g, 1M kcl 1ml, 1MMgso 4 4ml, 1M MgCl 4ml, 1M NaCl 4ml, and finally make up to 400ml. Adjust the pH to 7.0. After high-pressure steam sterilization, 4ml of 4M glucose (Glucose) was added to the cooling solution.

[0070] 1.2.3X-gal: 100mg dissolved in 2ml dimethylformamide, sterilized by filtration with a 0.22um filter membrane, and stored at -20°C.

[0071] 1.2.4IPTG: 1.2g IPTG, add deionized sterilized water to a final volume of 50ul, filter with a 0.22um membrane filter to sterilize.

[0072] 1.2.5Amp: 100mg Amp, add 1ml deionized sterilized water, filter and steril...

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Abstract

The method for detecting that LRP6 is the target gene of miR-29a by using a dual-luciferase reporter gene, the purpose of the present invention is to confirm that miR-29a can be negatively expressed by targeting the 3'UTR of the LRP6 gene through a dual-luciferase reporter gene system Regulates the expression of LRP6. A method for detecting LRP6 as a target gene of miR-29a using a dual-luciferase reporter gene, the method comprising the following steps: (1) constructing a plasmid vector and performing point mutations: (2) dual fluorescence detection. The present invention The beneficial effects are as follows: provide a new method for verifying that miR-29a can negatively regulate the expression of LRP6 by targeting the 3'UTR of the LRP6 gene, and contribute to miR-29a, LRP6 and Wnt / -catenin signaling pathways Elucidation of the pathogenesis of osteogenesis-related diseases involved; contribute to the development of potential drugs for osteogenesis-related diseases involved in miR‑29a, LRP6 and Wnt / ‑catenin signaling pathways and the screening of biomarkers.

Description

technical field [0001] The invention relates to a method for detecting LRP6 as a target gene of miR-29a by using a dual luciferase reporter gene. Background technique [0002] MicroRNAs (miRNAs) are small non-coding RNAs with a size of about 19 to 25 bases, which achieve gene expression by complementary matching with the 3' untranslated region (3'UTR) of the mRNA of the target gene Regulation plays an extremely important role in a series of physiological and pathological processes. Post-transcriptional repression of gene expression mediated by miRNAs plays an important role in the regulation of osteogenic differentiation. miR-29a is a positive regulator of osteogenic differentiation and controls collagen expression in differentiated osteoblasts. During the differentiation of mesenchymal cells into human osteoblasts, miR-29a is indispensable, and in osteoblasts, the expression of miR-29a is upregulated. [0003] Studies have shown that miR-29a can play different roles in d...

Claims

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Application Information

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IPC IPC(8): C12Q1/66
CPCC12Q1/66
Inventor 黄进贤
Owner THE UNIV OF HONG KONG SHENZHEN HOSPITAL
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