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Hepatoma diagnosis kit for detecting microRNA expression level and application thereof

A technology for detection reagents and diagnostic reagents, applied in the fields of biology and medical testing, can solve problems such as unreported liver cancer diagnostic kits, and achieve the effects of good clinical application value, strong clinical operability, and simple detection methods.

Inactive Publication Date: 2019-05-03
SHANGHAI HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are no reports about the liver cancer diagnostic kit and its application for detecting the expression level of microRNA.

Method used

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  • Hepatoma diagnosis kit for detecting microRNA expression level and application thereof
  • Hepatoma diagnosis kit for detecting microRNA expression level and application thereof
  • Hepatoma diagnosis kit for detecting microRNA expression level and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation of Example 1 Liver Cancer Diagnostic Kit

[0050] The production process and operation process of the diagnostic kit for detecting primary liver cancer patients are mainly based on PCR technology.

[0051] The expression level of microRNA in patients with primary liver cancer was screened by quantitative PCR and other techniques, and used as an index to predict the diagnosis of patients with primary liver cancer. The miR-125a-5p amplification primer pair sequence is shown in SEQID.2 and SEQID.3 (see Table 1), and the internal reference primer pair is shown in SEQID.4 and SEQID.5 (see Table 1).

Embodiment 2

[0052] Example 2 miRNA gene chip analysis

[0053] 1. Experimental method

[0054] 1. Cell culture: Human liver cancer cell lines Bel-7404, SK-Hep1, Bel-7402, MHCC-97H and normal liver cell L02 were obtained from American Type Culture Collection (ATCC, MD, USA). Liver cancer cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 5*105 per well, and all these cells were treated with 10% fetal bovine serum (FBS, Servicebio, G8001-500, China) and 1% The high glucose medium (DMEM, Sigma, RNBG6034, USA) of penicillin-streptomycin (KeyGen, KGY0023, China) was cultured in a 37°C incubator with 5% CO2.

[0055] 2. miRNA gene chip analysis

[0056] The miRNA gene chip analysis was carried out according to the routine operation method.

[0057] 2. Experimental results

[0058] Gene chip results such as figure 1 , The analysis results screened out the miRNA with difference——miR-125a-5p.

Embodiment 3

[0060] 1. Experimental method

[0061] 1. Cell culture: Human liver cancer cell lines Bel-7404, SK-Hep1, Bel-7402, MHCC-97H and normal liver cell L02 were obtained from American Type Culture Collection (ATCC, MD, USA). Liver cancer cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 5*105 per well, and all these cells were treated with 10% fetal bovine serum (FBS, Servicebio, G8001-500, China) and 1% Penicillin-streptomycin (KeyGen, KGY0023, China) was cultured in high glucose medium (DMEM, Sigma, RNBG6034, USA) in 5% CO 2 in a 37°C incubator.

[0062] 2. Extraction of total cellular RNA: centrifuge the cells obtained in the previous step at 1000×rpm for 5 minutes, collect the cells into 1.5ml centrifuge tubes, add 1ml Trizol (Sigma, MKCB9720, USA) to each tube, and gently Mix by pipetting, let stand at room temperature for 5 minutes, add 200 μl of chloroform to each tube, shake and mix vigorously for 30 seconds, and centrifuge at 12000×g ...

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Abstract

The invention belongs to the technical field of biological and medical examination, and particularly relates to a kit using the microRNA expression level for diagnosing hepatoma and application of thekit. Firstly, miRNA gene chip analysis is performed to discover different miRNA-miR-125a-5p; then, RNA extraction, reverse transcription and real-time polymerase chain reaction (RT-qPCR) are performed to obtain the expression quantity of miR-125a-5p, which proves that the miR-125a-5p is lower expressed in a hepatoma cell line than that of a normal liver cell. On this basis, the invention developsthe kit and method for detecting the expression of miR-125a-5p. The kit has the advantages of having a good diagnosing value in hepatoma diagnosis and AFP low-expressed hepatoma, and the survival chance is greatly increased for hepatoma patients.

Description

technical field [0001] The invention relates to the technical field of biology and medical examination, in particular to a liver cancer diagnostic kit for detecting the expression level of microRNA and its application. Background technique [0002] Primary liver cancer is one of the malignant tumors with high morbidity and mortality in my country. The latest global cancer statistics show that the global incidence of primary liver cancer ranks sixth, while the mortality rate ranks fourth (Bray, F. et al. (2018) CA: AC Cancer Journal for Clinicians). In my country, its incidence rate ranks third among malignant tumors, and its mortality rate ranks second (Chen Wanqing, et al. (2017) China Tumor 26(1), 1-7). In the early stage of primary liver cancer, surgery, liver transplantation and local interventional therapy can greatly improve the patient's survival rate (Tunissiolli, N.M.etal. (2017) AsianPacJCancerPrev18, 863-72), but also have to face the 5-year recurrence rate as hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/113
Inventor 马丽芳徐鑫牛永杰张聪聪陶玉泉汪智娴姜鸿圆徐晋豫蔡枫
Owner SHANGHAI HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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