9 microRNA markers for predicting whether primary liver cancer relapses

A primary liver cancer, sequence technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, the use of vectors to introduce foreign genetic material, etc., can solve the problem that the role cannot be underestimated

Inactive Publication Date: 2011-04-06
SHANGHAI INST OF ONCOLOGY
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  • Abstract
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Problems solved by technology

[0006] Although the proportion of microRNA in the total RNA of cells is very small, because it can efficiently regulate all mRNAs with target sites, the role of microRNA in the development of organisms and even the occurrence and development of tumors is still important. Not to be underestimated

Method used

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  • 9 microRNA markers for predicting whether primary liver cancer relapses

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preparation example Construction

[0074] The preparation of the miRNA chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The miRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and then fixing it by standing overnight. If the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-radioactive Operation Manual" edited by Wang Shenwu; J.L.erisi, V.R.Iyer, P.O.BROWN. Exploring the metabolic and genetic control of gene expression on agenomic scale.Science, 1997; 278:680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.

[0075...

Embodiment 1

[0094] The preparation of embodiment 1 RNA sample

[0095] Tissue sample:

[0096] 23 pairs of cancer and paracancerous tissues (including 10 samples with recurrence and 13 samples without recurrence) were obtained from surgical resection specimens of patients with primary liver cancer (HCC). These specimens were obtained from the First Affiliated Hospital of Zhejiang University and Qidong Liver Cancer Research Place. All the above-mentioned specimens were obtained with the approval of the ethics committee of the WHO partner organization authorized by the Shanghai Municipal Government. The clinical data of tissue samples include: gender, age, tumor size, pathological grade (Edmonson), liver cirrhosis, metastasis, recurrence, HBV and HCV infection, etc. All patients did not receive chemotherapy before operation, and the postoperative follow-up was up to 60 months.

[0097] Gene chip:

[0098] The microRNA expression profiling chip adopts the miRNA expression profiling chip ...

Embodiment 2

[0104] Extraction and labeling of embodiment 2microRNA

[0105] The miRNAs were extracted with Ambion's miRNAs extraction kit, and the specific operations were performed according to the corresponding instructions. Samples were labeled with T4 RNA ligase according to Thomson's method. In short, here's how:

[0106] 1.4 μg small RNA and 500ng 5'-phosphate-cytosine-uracil-cy3-3' (Dharmacon, Chicago, USA) and 2 units of 2units T4 RNA ligase (NEB, Ipswich, USA), incubated at 4°C for 2 hours , to label the miRNA. An equal amount of corresponding negative control was set up for each miRNA sample.

[0107] 2. The labeled RNA was precipitated with 0.3M sodium acetate and 2.5 volumes of ethanol, and then resuspended with 15 μl of hybridization solution containing 3×SSC, 0.2% SDS and 15% formamide. All hybridizations were repeated twice, and the hybridization was performed with LifterSlip TM (Erie, PA USA) to ensure that the hybridization solution flows evenly between the chip and t...

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Abstract

The invention relates to 9 microRNA markers for predicting whether the primary liver cancer relapses, in particular to a kind of microRNA markers for predicting whether the primary liver cancer relapses. The tests prove that the specific microRNA markers can very effectively distinguish the samples with relapse and the samples without relapse of the primary liver cancer. The invention also relates to a chip and a kit for detecting the microRNA markers.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, the present invention relates to a class of microRNA markers that can be used to predict whether primary liver cancer relapses and its use. The invention also relates to a chip and a kit for detecting the microRNA marker. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in my country, accounting for about 50% of new cases in the world every year. Liver cancer is highly malignant, develops rapidly, is difficult to treat, and has a high mortality rate. Therefore, the early diagnosis of liver cancer becomes more and more urgent. [0003] The occurrence and development of anything is under the joint action of internal and external factors, internal factors are dominant, and external factors are supplementary. As the genetic medium of life, genes are the basic internal causes in the birth, aging, disease and death of organisms. Mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12Q1/68C40B40/06
Inventor 何祥火魏霖梁琳慧顾健人
Owner SHANGHAI INST OF ONCOLOGY
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