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Application of miRNA-199a in preparation of diagnostic kit

A technology of miRNA-199a and diagnostic kits, applied in the fields of genetic engineering and medical diagnosis, can solve the problems of unsatisfactory sensitivity and specificity, and achieve improved sensitivity and specificity, easy detection, and accurate quantification Effect

Inactive Publication Date: 2014-11-19
FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity and specificity of these two methods are not very satisfactory

Method used

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  • Application of miRNA-199a in preparation of diagnostic kit
  • Application of miRNA-199a in preparation of diagnostic kit
  • Application of miRNA-199a in preparation of diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Screening of miRNAs associated with human liver cancer

[0056] 1.1 Collection of samples and collation of sample data

[0057] The inventor collected a large number of peripheral blood samples from patients with liver cancer and benign liver diseases in Beijing Cancer Hospital in January 2013 (the samples used for research were collected at the same time, sampled, subpackaged, and stored under uniform conditions). The inventor selected 20 cases of plasma from patients with liver cancer (hepatitis B-cirrhosis-liver cancer) randomly collected in the same hospital and as far as possible in the same department, and 20 cases of plasma from patients with benign liver diseases randomly collected in the same hospital and the same period as the experimental group. Try to avoid taking plasma samples from people with a family history of liver cancer for miRNA chip detection.

[0058] 1.2 miRNA microarray detection

[0059] 1.2.1 Isolation and purification of exosome ...

Embodiment 2

[0076] Example 2 QPCR verification of differentially expressed miRNA-199a

[0077] According to the detection results of the miRNA chip, miRNA-199a was selected for large sample QPCR verification. According to the method of sample collection and sample data arrangement in Example 1, 100 samples were selected from the liver cancer group and the benign liver disease group.

[0078] 2.1 The RNA extraction process is the same as in Example 1.

[0079] 2.2 Reverse transcription: mix 10pg-1μg total RNA template with 2μl 10* buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl ribonuclease (RNase) inhibitor and ribonuclease free water (RNase free water ) were mixed, the final volume was 20 μl, and incubated at 37° C. for 1 h. Then add 1 μl 0.5 μg / μl Oligo(dT) specific RT primer to the reaction tube, incubate at 70°C for 5 minutes and immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers. Finally, mix the above 20 μl reaction mixtu...

Embodiment 3

[0081] Example 3 Analyzing the diagnosis of miRNA on the pathogenesis of liver cancer

[0082]The exosome-derived miRNA-199a was used as the research object, and the sensitivity and specificity of the plasma free miRNA were compared in the control group and the liver cancer group. Taking liver cancer patients as the experimental group, liver cirrhosis patients and normal people as the control group, exoquic reagent was used to extract the exosome in the plasma (the steps are the same as in Example 1). 40 cases of liver cancer plasma samples, 20 cases of liver cirrhosis patients and 20 cases of normal human plasma samples were selected for miRNA-199a QPCR experiment, the CT value of each sample was obtained, and the scatter plot was made by using the ratio of sample miRNA-199a CT value to U6 CT value (Fig. 2), the experiment proved that the aggregation of exosome group was better than that of plasma group. The experimental results of exosome group were the same as the expressio...

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Abstract

The invention discloses a blood miRNA marker related to human liver cancer and an application. The marker is miRNA-199a from exosome. According to the invention, tumor-induced exosome in peripheral blood of liver cancer patient and liver benign disease patient can be separated and purified, miRNA-199a for expressing difference can be analyzed and screened by a miRNA chip, and the above conclusion is verified through QPCR. The marker and its detection reagent can be used for preparing a diagnostic kit and used for early diagnosis of liver cancer.

Description

technical field [0001] The invention belongs to the field of genetic engineering and medical diagnosis, and specifically relates to the use of miRNA, more specifically to the use of miRNA-199a in diagnosing liver cancer. Background technique [0002] Liver cancer is the fifth most common cancer and the third deadliest cancer in the world. 80% of liver cancers are often associated with chronic hepatitis B virus and / or hepatitis C virus infection. Such a high fatality rate is due to the lack of sensitive and specific non-invasive indicators for early diagnosis. Because the symptoms of early liver cancer are not obvious, it is usually diagnosed in the late stage. At that time, due to the intrahepatic and / or extrahepatic metastasis of cancer cells, the patient has lost the opportunity for surgical resection of the lesion. At present, the diagnosis of liver cancer mainly relies on the detection of intrahepatic tumors on imaging, including ultrasound, CT, nuclear magnetic resona...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 肖文华王硕屈雪玲李小梅董伟伟赵慧霞
Owner FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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