- In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.
NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.
NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.
- Prepare the following solutions:
NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.
Solution A: For each transfection, dilute 10 μl of resuspended shRNA Plasmid DNA (i.e. 1 μg shRNA Plasmid DNA) into 90 μl shRNA Plasmid Transfection Medium: sc-108062.
Solution B: For each transfection, dilute 1 - 6 μl of shRNA Plasmid Transfection Reagent: sc-108061 with enough shRNA Plasmid Transfection Medium: sc-108062 to bring final volume to 100 μl.
NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium: sc-108062.
NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.
NOTE: Although highly efficient in a variety of cell lines, shRNA Plasmid Transfection Reagent: sc-108061 may not be suitable for use with all cell lines.
- Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.
- Wash the cells twice with 2 ml of shRNA Transfection Medium: sc-108062 Aspirate the medium and proceed immediately to the next step.
NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.
- For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
- Add the 200 μl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer.
- Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
- Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the cells. Longer transfection times may be desirable depending on the cell line.
- Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).
- Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.
OPTIONAL: For transient transfection, aspirate media and replace with fresh1x normal growth medium. Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the previous step.
For selection of stably transfected cells, proceed with puromycin selection as follows:
NOTE: The working puromycin concentration for mammalian cell lines ranges from 1-10 μg/ml. Prior to using the puromycin antibiotic (sc-108071), titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
48 hours post-transfection, aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA. Control shRNA Plasmids include: sc-108060, sc-108065 and sc-108066.
NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Sample Buffer (sc-24945) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.
NOTE: For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.
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shRNA Plasmid DNA Mediated Inhibition of Gene Expression |
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