Application of lncRNA IGFL2-AS1 as a diagnostic marker for colon cancer
A technique for colon cancer, biological samples, applied in the field of tumor molecular biology
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Embodiment 1
[0031] Example 1: Real-time PCR detection of lncRNA IGFL2-AS1
[0032] Extract total RNA:
[0033] From colon cancer tissue samples and adjacent tissue samples, add 1 mL After the Reagent was placed at room temperature (15-30°C), the nucleoprotein complexes were completely separated. After 5 min, add 0.2 mL of chloroform, cover the tube, shake vigorously by hand for 15 s, and leave at room temperature for 2-3 min. Centrifuge at 12 000 g for 15 min at 2-8 °C, and transfer the upper colorless aqueous phase to another 1.5 mL Eppendorf tube. Add 0.5 mL of isopropanol and leave at room temperature for 10 min. Centrifuge at 12 000 g for 10 min at 2-8 °C and discard the supernatant. The RNA pellet was washed with 1 mL of 75% ethanol and mixed with a vortex mixer. Centrifuge at 7500g for 5 min at 2-8°C and discard the supernatant. Use RNase-free ddH after air drying for 5-10 min 2 O dissolves RNA, blows and sucks repeatedly with a micropipette and incubates at 55-60 °C for 10 ...
Embodiment example 2
[0044] Example 2: Knockdown of lncRNA IGFL2-AS1 in cancer cells
[0045] The SW620 cell line was selected for the knockdown experiment of lncRNA IGFL2-AS1, and the related gene expression and cell migration ability after knockdown were detected.
[0046] Knockdown of lncRNA IGFL2-AS1:
[0047] The knockdown of lncRNA IGFL2-AS1 was achieved by siRNA. The specific method was as follows: cells were plated on a six-well plate, and when the plate was 50% full the next day, starve with Opti-MEM for 30 minutes;
[0048] Dissolve 2.5 nmol siRNA / NC with 125 μL of DEPC water, and the amount of each well is 10 μL. Take two tubes labeled A and B, add 100 μL Opti-MEM to each tube; add 10 μL lipofectamine 3000 to tube A, add 10 μL siRNA or NC to tube B, mix well and let stand at room temperature for 5 min. The two tubes of AB were fully mixed, and added to the six-well plate for 15 min, and transfected for 6-8 h.
[0049] The siRNA used in the research of the present invention, the seque...
Embodiment example 3
[0057] Implementation Case 3: CCK-8 Experiment
[0058] Take 1×10 5 SW620 cells were seeded in a 12-well plate at 37°C with 5% CO. 2 Culture, when the cells grow to 70% confluence the next day, remove the original medium, add cells, trypsinize after 24 h, and inoculate 3 000 cells / well in a 96-well plate, with 5 duplicate wells in each group . After culturing for 1-7 days, 10 μl of CCK-8 was added to each well, and the culture was continued for 1 h. The blank well was used to adjust zero, the wavelength was 450 nm, and the OD value was detected by an automatic enzyme label reader.
[0059] The result is as image 3 shown, the cells transfected with siRNA had reduced proliferative capacity compared to tumor cells that were not knocked down.
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