A molecular marker for triple-negative breast cancer and its application
A specific technology for triple-negative breast cancer, applied in the biological field, can solve problems such as unclear pathogenesis, and achieve the effect of enhancing migration and invasion capabilities
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Embodiment 1
[0033] Example 1 Cell culture, transfection and Western Blot detection of the interference effect of RNF187 protein in cell lines
[0034] Cell recovery: Take out the MDA-MB231 cells from the -80°C refrigerator, and immediately put them into a 37°C water bath for rapid thawing, so that the cells can be completely thawed in the shortest time. After wiping and disinfecting the surface of the freezing tube with 75% alcohol, it was transferred to a 5 mL EP tube prepared in advance (1 mL DMEM+10% FBS had been added in advance), and centrifuged at 900 rpm for 3 min. Discard the supernatant, resuspend with 1mL DMEM complete medium (DMEM+10%FBS), mix well, add 5mL DMEM complete medium (DMEM+10%FBS) to the culture flask, transfer the resuspended cell suspension to culture flasks at 37°C, 5% CO 2 Culture in an incubator, replace the medium the next day, continue to culture, and start passage when the cell density is about 80%-90%.
[0035]Cell subculture: discard the old culture mediu...
Embodiment 2
[0040] Example 2 Detection of the mRNA interference effect of RNF187 in cell lines by real-time quantitative PCR
[0041] Cell subculture: discard the old culture medium in the culture flask with a cell density of about 80%-90%, rinse with pre-warmed PBS for 1-2 times, and then add trypsin digestion solution (0.25% trypsin) to the culture flask. +0.02%EDTA) 1mL, digested at 37℃ for 2-3min, observe the cell state under the microscope, when the cell volume shrinks and becomes round and the gap becomes larger, add 1mL DMEM complete medium (DMEM+10%FBS) to stop the digestion, and use a pipette to stop the digestion. The liquid gun gently and repeatedly blows the bottom of the bottle. Collect the pipetted cell suspension into a 5mL centrifuge tube at 900rpm per minute, centrifuge for 3min, discard the supernatant, resuspend with 1mL DMEM complete medium (DMEM+10%FBS), mix well, take 5mL DMEM complete medium (DMEM+10% FBS) was added to a new culture flask, and an appropriate amount...
Embodiment 3
[0049] Example 3 Using scratch test to detect the effect of triple negative breast cancer cell line migration ability after transfection
[0050]Cell subculture: discard the old culture medium in the culture flask with a cell density of about 80%-90%, rinse with pre-warmed PBS for 1-2 times, and then add trypsin digestion solution (0.25% trypsin) to the culture flask. +0.02%EDTA) 1mL, digested at 37℃ for 2-3min, observe the cell state under the microscope, when the cell volume shrinks and becomes round and the gap becomes larger, add 1mL DMEM complete medium (DMEM+10%FBS) to stop the digestion, and use a pipette to stop the digestion. The liquid gun gently and repeatedly blows the bottom of the bottle. Collect the pipetted cell suspension into a 5mL centrifuge tube at 900rpm per minute, centrifuge for 3min, discard the supernatant, resuspend with 1mL DMEM complete medium (DMEM+10%FBS), mix well, take 5mL DMEM complete medium (DMEM+10%FBS) into a new culture flask, transfer an...
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