Liver cancer diagnostic markers and diagnostic kits composed of serum microRNA

A kit, hsa-mir-192 technology, applied in the field of croRNA, can solve the problem of failing to clarify the value of early warning of circulating microRNA liver cancer, unable to comprehensively and objectively reflect circulating microRNA, and difficult to explain that markers are specific diagnostic markers for liver cancer, etc. problems, to achieve the effect of saving time and labor costs, avoiding complicated detection in the past, and mature experimental methods

Active Publication Date: 2016-08-31
江苏阔然生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the current research on circulating microRNA as a diagnostic marker for liver cancer still has the following deficiencies: 1) Most of the studies only selected microRNAs that were reported to be dysregulated in liver cancer tissues as candidate indicators, which may not be able to fully and objectively reflect the situation of circulating microRNA; 2) The sample size of some studies is small and lacks multi-center validation; 3) The control settings are not perfect, and it is difficult to explain that the identified markers are specific diagnostic markers for liver cancer; 4) All current studies are carried out in specimens that have been diagnosed with liver cancer However, there is a lack of prospective studies in high-risk groups, and the value of circulating microRNA for early warning of liver cancer has not been clarified

Method used

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  • Liver cancer diagnostic markers and diagnostic kits composed of serum microRNA
  • Liver cancer diagnostic markers and diagnostic kits composed of serum microRNA
  • Liver cancer diagnostic markers and diagnostic kits composed of serum microRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Collection and preparation of serum samples

[0033] The inventor collected healthy people (HC), hepatitis B carriers (IHC), chronic hepatitis B patients (CHB), liver cirrhosis patients (LC) and liver cancer patients (HCC) from August 2009 to August 2014. These populations met the inclusion criteria (Table 1), and according to the principle of gender and age matching, liver cancer and its control group specimens were set.

[0034] Training group: 257 serum samples collected from healthy people (51 cases), chronic hepatitis B patients (51 cases), liver cirrhosis patients (47 cases) and liver cancer patients (108 cases) from August 2009 to March 2012 .

[0035] Validation group 1: 352 serum samples from healthy people (60 cases), chronic hepatitis B patients (68 cases), liver cirrhosis patients (71 cases) and liver cancer patients (153 cases) collected from April 2012 to April 2013 specimen.

[0036] Validation group 2: 139 serum samples from healthy people ...

Embodiment 2

[0050] Example 2: qPCR Array and its data analysis

[0051] The inventor selected serum samples from 6 patients with liver cancer before operation and 8 patients with chronic hepatitis B at least one year before the last examination for qPCR Array screening. These patients were all male, and there was no significant difference in mean age and distribution (Table 4).

[0052] Table 4. Clinicopathological characteristics of specimens used for qPCR Array analysis

[0053]

[0054] The present invention adopts Applied Biosystems company's The Array Human MicroRNA method screens microRNAs that differ between liver cancer and chronic hepatitis B, and detects the levels of 754 known human microRNAs. See the Applied Biosystems website for specific steps. After the obtained raw data were calibrated, the inventors used Significant Analysis of Microarray (SAM) analysis method to select differential microRNAs, and finally screened and obtained 19 candidate microRNAs for subsequent ...

Embodiment 3

[0058] Embodiment 3: Real-time fluorescent quantitative PCR detects the microRNAs level of training group sample

[0059] 1.1 Extraction of serum RNA

[0060] The present invention uses Trizol reagent to extract, and obtains serum RNA through the method of phenol / chloroform extraction and purification, isopropanol precipitation, and glycogen-assisted precipitation, and the specific steps are as follows:

[0061] 1. Take 200 μl of serum, preferably add an equal volume of double-stranded RNA mixed with cel-miR-67 (NC67, based on the mature body sequence of nematode miR-67 that has no homology with the human genome sequence, and the final concentration is 0.2 nM, the sequence is shown in Table 5) of Trizol lysate, fully vortexed and mixed, and ice-bathed for 15 minutes.

[0062] 2. Add 100 μl of pre-cooled chloroform, shake and mix well, and centrifuge at 12,000 g for 15 min at 4°C.

[0063] 3. Transfer the supernatant, add an equal volume of pre-cooled phenol / chloroform (1:1),...

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Abstract

A hepatocellular carcinoma diagnostic marker consisting of serum mircoRNA is disclosed. The marker consists of hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, and hsa-miR-505. A hepatocellular carcinoma diagnostic kit is also disclosed. The kit contains a test for detecting the level of the described seven microRNA molecules in serum.

Description

technical field [0001] The present invention relates to a liver cancer diagnosis combination composed of serum microRNA, said serum microRNA includes hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa - miR-192 and hsa-miR-505, specifically related to the application of a kit for early warning and early diagnosis of primary hepatocellular carcinoma, belonging to the field of biomedical diagnosis. Background technique [0002] Primary liver cancer is a common malignant tumor with high morbidity and high lethality worldwide, among which hepatocellular carcinoma (referred to as liver cancer, Hepatocellular Carcinoma, HCC) accounts for more than 90% of primary liver cancer. According to the "Global Cancer Report 2014" issued by the World Health Organization, in 2012, the new cases of liver cancer in the world ranked fifth among malignant tumors, and its fatality rate ranked second. Among them, the new cases and deaths of liver cancer in China were about accou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/113
CPCC12Q1/68
Inventor 庄诗美崇雨田林雪嘉郭智伟元云飞杨晓静曾春贤方坚鸿
Owner 江苏阔然生物医药科技有限公司
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