Application of biomarker related to breast cancer occurrence and development
A technology for breast cancer and use, applied in the field of biomarkers, can solve the problems of unclear and rare relationship between expression status, lymph node metastasis and prognosis
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Embodiment 1
[0068] Example 1 Screening for gene markers associated with breast cancer
[0069] 1. Sample collection
[0070] The cancer tissue and corresponding normal tissue samples (5 cm from the tumor edge) of 4 cases of Luminal A breast cancer were collected for high-throughput sequencing. All patients did not receive chemotherapy, radiotherapy and endocrine therapy before operation, and all patients were informed Agree, all the above specimens were obtained with the consent of the organizational ethics committee, and the patient information is shown in Table 1.
[0071] Table 1 Sample information
[0072]
[0073] 2. RNA sample preparation and quality analysis
[0074] Use the Takara RNA Extraction Kit (Code NO.9767) to extract RNA from the tissue, the steps are as follows:
[0075] 1) Quickly transfer fresh or cryopreserved animal tissue samples to a liquid nitrogen pre-cooled mortar, grind the tissue with a pestle, and continuously add liquid nitrogen until it is ground into ...
Embodiment 2
[0110] Example 2 QPCR sequencing to verify the differential expression of the LINC01612 gene
[0111] 1. The differential expression of the LINC01612 gene was verified by large sample QPCR of the cancer tissue samples and normal tissue samples collected from 25 patients with Luminal A breast cancer according to the collection method in Example 1.
[0112] 2. RNA extraction
[0113] Takara RNA Extraction Kit (Code NO.9767) was used to extract RNA from tissue, see Example 1 for specific steps.
[0114] 3. QPCR
[0115] Primers were designed according to the gene sequences of LINC01612 and GADPH, and the primer sequences are shown in Table 3.
[0116] Table 3 Amplification primers
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[0118]
[0119] Using TaKaRa One Step TB Green TM Prime Script TM The RT-PCR kit (Code No. RR066A) was used for PCR reaction, and the reaction system and reaction conditions are shown in Table 4. In Thermal Cycler PCR amplification was carried out on the Time System amplificat...
Embodiment 3
[0124] Example 3 Expression of LINC01612 in breast cancer cell lines
[0125] 1. Cell culture
[0126] Culture the MCF-7 cell line of Luminal A breast cancer in 5% CO 2 , cultured in a constant temperature incubator at 37°C, and all cell culture media were added with 10% fetal bovine serum and 1% P / S. Change the medium once every 2-3 days, use 0.25% EDTA-containing trypsin for routine digestion, and passage the cells at a ratio of 1:3.
[0127] 2. Transfection
[0128] The LINC01612 overexpression vector and negative control plasmid were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd., using Lipofectamin from Invitrogen TM 2000 reagents were used for cell transfection, and the LINC01612 overexpression vector (experimental group) and negative control plasmid (control group) were transfected respectively.
[0129] Preparation before cell transfection Take the cells planted in the 6-well plate in advance in the incubator, wash the transfected cells twice with...
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