Molecular biomarker for diagnosing and treating triple-negative breast cancer and application thereof
A technology for triple-negative breast cancer and breast cancer cells, which is applied in the fields of crude drug medicine and molecular biology, and can solve the problem that the function and role of circRNA are not fully understood.
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Embodiment 1
[0147] 1. Sample collection
[0148] Collect 100 pairs of tumor tissues and normal control tissues from patients undergoing breast cancer surgery in Qilu Hospital of Shandong University from 2015 to 2019, and sign consent forms for all patients participating in the study.
[0149] 2. circRNA quantitative PCR experiment:
[0150] 2.1 Extraction of tissue total RNA
[0151] Total RNA was extracted from the tissues of breast cancer patients using Trizol reagent, and the concentration and purity of RNA were detected using corresponding equipment.
[0152] 2.2 Reverse transcription of total RNA
[0153] Reverse transcription was performed using the reverse transcription kit from Takara Company, the initial amount of RNA was 1 μl, and the reverse transcription of RNA was performed according to the instructions provided.
[0154] The system is shown in the table below:
[0155]
[0156] Specific reaction program settings in the PCR instrument:
[0157]
[0158] 2.3circRNA ...
Embodiment 2
[0168] At the same time, the present invention conducts statistics on the expression of circEIF3H in the sampled patients with triple-negative breast cancer, and conducts a comprehensive statistical analysis in combination with its therapeutic effect, such as Figure 4 As shown, it was found that triple-negative breast cancer patients with high expression of circEIF3H had a worse prognosis than triple-negative breast cancer patients with low expression of circEIF3H.
Embodiment 3
[0170] Effects on the growth rate and migration ability of cancer cells.
[0171] 1. The effect of circEIF3H on the growth rate of breast cancer cells
[0172] 1.1 Cell transfection
[0173] Take a 6cm petri dish as an example:
[0174] (1) Select a suitable cell culture dish according to the experimental requirements, count the cells one day before transfection, and plate according to the transfection requirements.
[0175] (2) Before transfection, replace the cell culture medium with an appropriate amount of medium without antibiotics, for example, add 2ml of medium to a 6cm dish.
[0176] (3) Take out the EP tube and mark the name of lipo or transfected siRNA respectively.
[0177] siRNA sequence:
[0178] si-circEIF3H-1: CAGCAGUCCAAUAUCAGAU (SEQ ID No. 6);
[0179] si-circEIF3H-2: CAGCCUUGCCAGCAGUCCA (SEQ ID No. 7).
[0180] (4) According to the ratio recommended in the instruction manual of Lipofectamine2000, take a 6cm dish as an example, take 250μl of opti-MEM to ...
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