A biomarker for the diagnosis and treatment of breast cancer
A breast cancer and material technology, applied in the field of biomarkers for the diagnosis and treatment of breast cancer, can solve the problems of breast cancer patients who cannot be treated
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Embodiment 1
[0047] Example 1 Screening for gene markers associated with breast cancer
[0048] 1. Sample collection
[0049] 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 or endocrine therapy before operation.
[0050] 2. RNA sample preparation and quality analysis
[0051] Use the Takara RNA Extraction Kit (Code NO.9767) to extract RNA from the tissue, the steps are as follows:
[0052] 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 powder. Add the powdered sample into a 1.5ml sterilized centrifuge tube containing Lysis Buffer RL, and pipette repeatedly until there is no obvious precipitation in the lysate.
[0053] 2) Centrifuge the lysate at 12,000 rpm at 4°...
Embodiment 2
[0087] Example 2 QPCR sequencing to verify the differential expression of the LINC02343 gene
[0088] 1. The differential expression of the LINC02343 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.
[0089] 2. RNA extraction
[0090] Takara RNA Extraction Kit (Code NO.9767) was used to extract RNA from tissue, see Example 1 for specific steps.
[0091] 3. QPCR
[0092] Primers were designed according to the gene sequences of LINC02343 and GADPH, and the primer sequences are shown in Table 2.
[0093] Table 2 Amplification Primers
[0094]
[0095] 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 3. In Thermal Cycler Perform PCR amplification on the RealTime System amplification instrument, ...
Embodiment 3
[0100] Example 3 Expression of LINC02343 in breast cancer cell lines
[0101] 1. Cell culture
[0102] 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.
[0103] 2. siRNA sequence
[0104] The siRNA-NC and siRNA-LINC02343 used in this application were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., and the sequence of silencing LINC02343 is shown in Table 4.
[0105] Table 4 siRNA sequence
[0106]
[0107] 3. Transfection
[0108] Using Lipofectamin from Invitrogen TM 2000 reagent for cell transfection, the experiment was divided into three groups: control group (MCF-7), negative control group (siRNA-NC) and experimental group (siRNA1, siRNA2, siRNA3), wherein the neg...
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