2.6 RNA pull-down assay
The RNA was first marked by Pierce Magnetic RNA-protein pull-down Kit (Thermo), and then the RNA precipitated overnight was removed and centrifuged at 4℃ for 30min at maximum speed. Add 20μL nuclease free-water to each tube to dissolve RNA. Take 400μ L beads from each tube, place them on a magnetic frame, and remove the supernatant. Wash with 800μ L 1X Binding & Washing buffer for 3 times and suck out the supernatant. Add 400μ L 2X Binding & washing buffer; Add 20μL RNA and 380μL DEPC water to make the final volume 800μL, Rotate the beads slowly at room temperature for 20min to fully combine with RNA. Add 500μ L cell lysate to each tube. Meanwhile, add RNase Inhibitor (1U/μL) to each tube. Rotate slowly at 4℃ for 2h to fully combine beads with cell lysis fluid. QPCR was performed after reverse transcription.
2.7 Plasmid DNA transfection and construction of overexpression vector .
One day before transfection, plate 0.5-2×105 cells in 500 ul of growth medium without antibiotics so that cells will be 90-95% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 4-8 ×105cells in 500ul of growth medium without antibiotics.For each transfection sample, prepare complexes as follows:a.Dilute DNA (pCDNA3.1-EGFP-T2A-Puro) in 50 ul of Opti-MEN Reduced Serum Medium without serum, mix gently.b.Mix Lipofectamine 3000 gently before use, then dilute the appropriate amount in 50 ul of Opti-MEN Medium. Incubate for 5 minutes at room temperature.c.After the 5 minutes incubation, combine the diluted DNA with diluted Lipofectamine 3000 (total volume=100ul).Mix gently and incubate for 20 minutes at room temperature.Complexes are stable for 6 hours at room temperature. Add the 100 ul of complexes to each well containing cells and medium.Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. Medium may be changed after 4-6 hours.Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium the following day.