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Use of serum/plasma microRNA in early diagnosis of HBV infection and liver cancer

A serum and plasma technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc.

Active Publication Date: 2013-04-10
MICROMEDMARK BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the diagnosis of HCC caused by HBV infection is limited to various biochemical indicators in serum / plasma, as well as traditional medical and imaging methods, and there is no report on the use of serum / plasma microRNA in the diagnosis and treatment of liver cancer

Method used

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  • Use of serum/plasma microRNA in early diagnosis of HBV infection and liver cancer
  • Use of serum/plasma microRNA in early diagnosis of HBV infection and liver cancer
  • Use of serum/plasma microRNA in early diagnosis of HBV infection and liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] The collection of embodiment 1 sample and the arrangement of sample data

[0111] The inventor collected a large number of serum samples from the First Affiliated Hospital of Nanjing Medical University and Nanjing Hospital of Infectious Diseases from September 2007 to July 2008. By sorting out the sample data, the inventor selected 80 cases Healthy controls (mean age: 37.52±12.09, age span: 20-75, male: 37, female: 43), 80 cases of viral clearance (mean age: 37.2±9.85, age span: 25-52, male: 48 , female: 32), 55 cases of long-term asymptomatic HBV infection (average age: 35.80±11.40, age span: 21-67, male: 33, female: 22), 80 cases of chronic hepatitis B patients (average age: 36.22±12.18 , age range: 20-86, male: 55, female: 25), 65 HBV-related HCC patients (mean age: 53.43±9.10, age range: 40-83, male: 59, female: 6) and 48 cases Chronic hepatitis C patients (mean age: 35.96±7.15, age span: 24-58, male: 39, female: 9) were used as experimental samples for Solexa sequ...

Embodiment 2

[0153] Example 2 Solexa sequencing experiment of microRNA in serum / plasma

[0154] Using Solexa sequencing technology to discover and prove that there are 88 differentially expressed microRNAs in the serum / plasma of 30 healthy controls and 30 chronic hepatitis B patients. The specific steps are:

[0155] (1) Collect serum / plasma from healthy controls and chronic hepatitis B patients

[0156] (2) Take 80-100ml of serum respectively and add an equal volume of Trizol Reagent

[0157] (3) Phase separation

[0158] Leave at room temperature for 15 minutes, then add chloroform at a volume ratio of 0.2ml chloroform / 1ml Trizol Reagent, shake vigorously for 15s, room temperature for 15 minutes, centrifuge at 12,000g, 4°C for 15 minutes

[0159] (4) Transfer the aqueous phase to a new 50ml centrifuge tube, and remove the protein phase in 3 steps of phenol / chloroform

[0160] (5) RNA precipitation

[0161] Transfer the aqueous phase to a new centrifuge tube, add isopropanol accordin...

Embodiment 3

[0179] Embodiment 3 The real-timePCR experiment of microRNA in serum including Solexa sample

[0180] Use the primers in Table 2 to carry out the sera of 30 cases of healthy controls (Solexa sequencing samples), 30 cases of virus clearance, 30 cases of asymptomatic HBV infection, 30 cases of chronic hepatitis B patients (Solexa sequencing samples) and 30 cases of chronic hepatitis C patients qRT-PCR detection of microRNA.

[0181] (1) Preparation of cDNA samples: a) Take 500ul serum; b) Add an equal volume of water-saturated phenol, shake and mix, centrifuge at 13200rpm at 4°C for 3 minutes, and take the supernatant; c) Supernatant + 1 / 2 volume (250ul ) Phenol + 1 / 2 volume (250ul) chloroform, shake and mix, centrifuge at 13,200rpm at 4°C for 3 minutes, and take the supernatant; Minutes, take supernatant as RNA sample; e) then obtain cDNA by RNA reverse transcription reaction. The reverse transcription reaction system included 4 μl 5×AMV buffer, 2 μl 10 mM each dNTP mixture (...

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Abstract

A microRNA combination used for distinguishing HCV-infected persons, HBV-infected persons and HBV-related liver cancer patients from normal persons as well as the use thereof in early diagnosis of HBV infection and HBV-related liver cancer are provided. The combination contains all the serum / plasma microRNAs which have different expression profiles between HCV-infected persons, HBV-infected persons, HBV-related liver cancer patients and normal persons. A kit containing the microRNAs of the combination used for early diagnosis of HBV infection and HBV-positive liver cancer as well as the preparation method thereof are further provided. The main advantages of the kit include high specificity and high sensitivity.

Description

technical field [0001] The present invention relates to the application research of serum / plasma microRNA (miRNA) in the early diagnosis and dynamic monitoring of HBV-positive liver cancer, in particular to the early diagnosis, curative effect evaluation, drug screening and drug efficacy evaluation of HBV-related diseases, especially liver cancer. aspects of application. Background technique [0002] Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors in the world. The International Cancer Research Center (IARC) estimated that in 2000, the global incidence of liver cancer was 564,000, and the death rate was 549,000. Liver cancer accounts for more than half of the global morbidity and mortality, and is one of the major cancers that seriously endanger the life and health of our people. The occurrence of liver cancer is a multi-factor and multi-stage process. Globally, chronic HBV and HCV infection are the main risk factors for p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/68
CPCC12Q1/706
Inventor 曾科张辰宇张峻峰李丽民
Owner MICROMEDMARK BIOTECH CO LTD