Clonogenic assay using siRNA

Clonogenic assay using siRNA (Protocol optimized by Tariq Khan, Project JRF, LSN-101)   

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Seed 500-1000 cells (depending upon growth and metastatic property) per well in 1mL media (DMEM or RPMI-1640 with 10% FBS plus 1X antibiotics) in 6 well plate. Incubate the cells for 24 hours at 37° C with 5% CO2. Serum starvation is given for 12 to 18 hours for cells synchronization (same phase of cell cycle) and efficient transfection (FBS in media hampers siRNA transfection). Note: Try to add Opti-MEM incomplete media (7pm) at night before leaving lab after 24 hours of incubation so that next morning (10am) cells will be ready for transfection. Prepare siRNA for transfection for 6 well plate (3 wells Control and 3 wells RhoG), label tubes (Tube A, A’ & B, B’ where A is siRNA, B is GC duplex, A’ & B’ are Lipofactamine). Add optimized dose (like 10µL RhoG siRNA, 50nM) of siRNA in tube A having 150µL Opti-MEM incomplete media & 8µL of Lipofactamine in tube B having 150µL. Incubate both tubes (A&B and A’&B’) separately for 15 minutes inside culture hood at 25° C. Mix both tubes (A in A’ and B in B’) again incubate for 15 minutes in biosafety cabinet. After incubation add 100µL per well in 3 wells of 6 well plate (3 Control & 3 siRNA) in same 1mL Opti-MEM incomplete media. Incubate transfected cells for 6 to 8 hours for transfection and a total of 24 hours for maximum transfection. Add 1% FBS after 8 hours of transfection. Note: Add 10µL of FBS in wells containing cells and having 1mL of Opti-MEM incomplete media. Incubate cells for colony formation for additional 3 days inside incubator at 37° C with 5% CO2. Observe cells daily under microscope for colony formation and contamination. A total of 7 days incubation of cells is required minimum for colony formation, 24 hours for cells growth after trypsinization, 12 to 18 hours serum starvation, 24 hours for transfection and 3 days incubation after transfection. Terminate the experiment on 7th day. Note: Cells after 24 hours of transfection add only 1% FBS in 1mL opti-MEM complete media. After 7 days discard the media, wash with PBS and 500µL of chilled absolute methanol each well to fix the cells. Keep the plate at -20°C for 1 hour. Stain with 0.25% giemsa or 0.4% trypan blue by adding 500µL of stain each well. Leave for 5 minutes and wash with PBS twice. Count number of colonies of more than 50 cells. Data represented as percent colonies.