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Application of miRNA-26a

A miRNA-26a, marker technology, applied in the field of use, can solve the problem of undiscovered miRNA functions, and achieve the effects of improving sensitivity and specificity, easy detection, and accurate quantification

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

AI Technical Summary

Problems solved by technology

Sequence analysis shows that more than 1 / 3 of human genes are directly regulated by miRNAs, and more and more experimental evidence shows that miRNAs still have many undiscovered functions

Method used

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  • Application of miRNA-26a
  • Application of miRNA-26a
  • Application of miRNA-26a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening of miRNAs associated with human liver cancer

[0052] 1.1 Collection of samples and collation of sample data

[0053] 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.

[0054] 1.2 miRNA microarray detection

[0055] 1.2.1 Isolation and purification of exosome ...

Embodiment 2

[0072] Example 2 QPCR verification of differentially expressed miRNA-26a

[0073] According to the detection results of the miRNA chip, miRNA-26a 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.

[0074] 2.1 The RNA extraction process is the same as in Example 1.

[0075] 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 mixture...

Embodiment 3

[0077] Example 3 Analyzing the diagnosis of miRNA on the pathogenesis of liver cancer

[0078]The exosome-derived miRNA-26a was used as the research object, and compared with plasma free miRNA in the sensitivity and specificity of the control group and 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-26a QPCR experiment, the CT value of each sample was obtained, and the scatter plot was made by using the ratio of sample miRNA-26a CT value to U6CT value (Figure 2) The experiment proved that the exosome group had better aggregation than the plasma group. The exosome group experimental results had the same expression trend of miRNA-26a in liver...

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Abstract

The invention discloses a blood miRNA marker related to human liver cancer and an application. The marker is miRNA-26a 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 in the liver cancer patient and liver benign disease patient can be analyzed and screened by a miRNA chip, and the liver cancer can be determined for the subjects by detecting miRNA-26a. 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, and more specifically relates to the use of miRNA-26a in diagnosing liver cancer. Background technique [0002] Primary hepatocellular carcinoma is a common malignant tumor of the digestive tract and ranks fifth in the world. About 6.35 million new cases of malignant tumors are discovered in the world every year, of which liver cancer accounts for about 260,000 cases and 260,000 cases 42.5% of them occur in our country and the incidence rate is still increasing year by year. Liver cancer is generally found at an advanced stage, and the effective treatment methods are liver resection and liver transplantation. Conventional chemotherapy has no obvious effect on the treatment of liver cancer. Therefore, early diagnosis of liver cancer is particularly important. Exploring effective clinical diagnostic markers and treatment strategi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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