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MicroRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue

A sequence and forward technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc.

Inactive Publication Date: 2013-01-02
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • MicroRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue

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preparation example Construction

[0075] 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 a genomic scale. Science, 1997; 278: 680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.

[0...

Embodiment 1

[0095] The preparation of embodiment 1 RNA sample

[0096] 1. Tissue samples:

[0097] Forty-six pairs of cancer and paracancerous tissues were obtained from surgical resection specimens of primary lung cancer patients, and these specimens were obtained from Shanghai Chest Hospital. 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 the tissue samples include: gender, age, tumor size, pathological grade (TNM stage), metastasis or recurrence, etc.

[0098] 2. Gene chip:

[0099] microRNA expression profiling chip, using the crystal core of Boao Biotechnology Co., Ltd. Expression profiling chip (single-channel chip).

[0100] 3. Extraction of total tissue RNA

[0101] 3.1 Collection and storage of samples: The isolated tissues were cut into small pieces and immediately frozen in liquid nitrogen, and then moved to a -80°C refrigerator ...

Embodiment 2

[0105] Example 2 Extraction and labeling of microRNA

[0106] 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:

[0107] 1.4 μg small RNA and 500ng 5′-phosphate-cytosine-uracil-cy3-3′ (Dharmacon, Chicago, USA) and 2 units of T4RNA ligase (NEB, Ipswich, USA) were incubated at 4°C for 2 hours. miRNAs are labeled. An equal amount of corresponding negative control was set up for each miRNA sample.

[0108] 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 the cove...

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Abstract

The invention relates to microRNA makers for discriminating constitutional lung cancer tissue and paracancerous tissue. More specifically, the invention relates to 38 microRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue. It has been proved by examination that, these specific microRNA markers can effectively discriminate constitutional lung cancer tissue and paracancerous tissue. The invention also relates to a chip and a test kit for detecting the microRNA markers.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to a class of microRNA markers that can be used to distinguish primary lung cancer from paracancerous tissues and uses thereof. The invention also relates to a chip and a kit for detecting the microRNA marker. Background technique [0002] Lung cancer is one of the most common malignant tumors in my country, with high degree of malignancy, rapid development, difficult treatment and high mortality. Therefore, the early diagnosis of lung 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. Most genes exert their biological functions by transcribing into ribo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12Q1/68
Inventor 何祥火梁琳慧陆舜陈智伟虞永峰
Owner SHANGHAI INST OF ONCOLOGY
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