Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome

Active Publication Date: 2015-07-15
CHILDRENS HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the proportion of microRNA in the total RNA of cells is very small, because it can efficiently regulate all mRNAs with target sites,

Method used

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  • Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome
  • Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome
  • Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The preparation of embodiment 1 RNA sample

[0077] Serum samples from 40 biliary atresia and 35 cholestatic infantile hepatitis syndrome were obtained from the Children's Hospital of Fudan University. All the above-mentioned specimens were obtained with the approval of the ethics committee of the WHO partner organization authorized by the Shanghai Municipal Government. Using mirVanaTM PARISTM (miRNA specially used for extracting blood) (Cat#AM1556, Ambion, Austin, TX, US) and according to the standard operating procedure provided by the manufacturer, the total RNA extraction of the sample was carried out, and the extracted total RNA was passed through Agilent Bioanalyzer2100 (Agilent technologies, Santa Clara, CA, US) After passing the electrophoresis quality inspection, it is ready for use. Gene chip: microRNA expression profile chip, using miRNA expression profile chip (single-channel chip) from Agilent Co., Ltd.

Embodiment 2

[0078] Extraction and labeling of embodiment 2microRNA

[0079] The miRNAs were extracted with Ambion's miRNAs extraction kit, and the specific operations were performed according to the corresponding instructions. Samples were labeled with T4 RNA ligase according to Thomson's method.

[0080] In short, here's how:

[0081] (1) Obtain total RNA from Example 1, then use Ambion's miRNAs extraction kit to extract miRNA; take out 5'-phosphate-cytosine-uracil-cy3-3' of 1.4ug miRNA and 500ng ( Dharmacon, Chicago, USA) and 2 units of T4RNA ligase (NEB, Ipswich, USA), incubated at 4°C for 2 hours to label miRNA. An equal amount of corresponding negative control was set up for each miRNA sample.

[0082] (2) The labeled RNA was precipitated with 0.3M sodium acetate and 2.5 volumes of ethanol, and then resuspended with 15u1 hybridization solution containing 3X SSC, 0.2% SDS and 15% formamide. All hybridizations were repeated twice, and the hybridization was performed with LifterSlip ...

Embodiment 3

[0084] Example 3 Quantitative PCR kit detects microRNA expression

[0085] (1) microRNA reverse transcription

[0086] Using ABI Reverse Transcription Kit (Applied Biosystems),

[0087] The reaction system is as follows:

[0088] 100mM dNTPs (with dTTP)0.2μl

[0089] MultiScribe TM Reverse Transcriptase, 50U / μL1μl

[0090] 10×Reverse Transcription Buffer1μl

[0091] RNase Inhibitor, 20U / μL0.13μl

[0092] RT Primer for microRNA (1μl each)

[0093] RNA (10ng / μl) 3μl

[0094] Water 1.67μl

[0095] Total10μl

[0096] (2) MicroRNA fluorescence quantitative PCR detection

[0097] Using TaqMan MicroRNA Kit (Applied Biosystems),

[0098] The reaction system is as follows:

[0099] TaqMan MicroRNA Assay (20×) 0.5μl (probe halved)

[0100] Template: 2ul diluent (stock solution diluted 10 times)

[0101] PCR Mix10ul

[0102] Water 7.5ul

[0103] Total20ul

[0104] The reaction conditions are as follows:

[0105] 95 degrees 5min

[0106] 95 degrees 10s40cycles

[0107]...

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Abstract

The invention belongs to the field of biological medicine and molecular biology, relates to a novel biliary atresia diagnostic marker, and more specifically provides applications of a set of microRNAs in preparing biliary atresia diagnostic preparations. The microRNA is composed of hsa-miR-150, hsa-miR-4689, hsa-miR-92a-3p, and hsa-miR-4429. It is confirmed by experiments that, in biliary atresia serum, expression amount of the miRNA possesses significant difference on the statistical significance with expression amount of a corresponding miRNA of cholestatic infant hepatitis syndrome. The invention also provides a detection method of the set of miRNAs. Compared with the prior art, the detection method is capable of saving time, and is high in sensitivity and accuracy, low in cost, and/or high in efficiency. And in addition, the invention also provides kits and chips relative to the applications.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, the present invention relates to the detection and application of a class of microRNA serum markers that can be used to distinguish between biliary atresia and cholestatic infantile hepatitis syndrome. The invention also relates to a chip and a kit for detecting the microRNA marker. Background technique [0002] Biliary atresia is a common malformation in pediatric surgery characterized by atresia of extrahepatic and extrahepatic bile ducts and obstructive jaundice, with an incidence rate of 1 / 5000-1 / 8000. It is a disease that occurs in children and is characterized by progressive sclerosing bile duct inflammation, obstruction and destruction of bile ducts at all levels, liver fibrosis and cirrhosis. Many children often have severe liver fibrosis when biliary atresia is diagnosed and soon develop cirrhosis, eventually leading to liver failure. Therefore, the early diagnos...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郑珊董瑞陈功
Owner CHILDRENS HOSPITAL OF FUDAN UNIV
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