Here we discuss a protocol forÂ shRNA Gene Silencing and CRISPR system transfection of which include puromycin.
shRNA Plasmid DNA Mediated Inhibition of Gene Expression
- In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.
NOTE:Â 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:Â 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:Â The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 Î¼g of shRNA Plasmid DNA and between 1.0 and 6.0 Î¼l 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 Î¼g:1 Î¼l, then amounts ranging from 0.5 Î¼g/0.5 Î¼l to 2.0 Î¼g/2.0 Î¼l should be tested.
Solution A: For each transfection, dilute 10 Î¼l of resuspended shRNA Plasmid DNA (i.e. 1 Î¼g shRNA Plasmid DNA) into 90 Î¼l shRNA Plasmid Transfection Medium.
Solution B: For each transfection, dilute 1 – 6 Î¼l of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring the final volume to 100 Î¼l.
NOTE:Â Do not add antibiotics to the shRNA Plasmid Transfection Medium.
NOTE:Â Optimal results may be achieved by using siliconized microcentrifuge tubes.
NOTE:Â Although highly efficient in a variety of cell lines, shRNA Plasmid Transfection Reagent 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 Aspirate the medium and proceed immediately to the next step.
NOTE:Â Do not use PBS; 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 Î¼l shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) using a pipette to the well, covering the entire layer.
- Gently mix by swirling the plate to ensure that the entire cell layer is immersed in the solution.
- Incubate the cells 5-7 hours at 37Â° 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.
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 a selection of stably transfected cells, proceed with puromycin selection as follows:
NOTE:Â The working puromycin concentration for mammalian cell lines ranges from 1-10 Î¼g/ml. Prior to using the puromycin antibiotic, 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:Â Controls should always be included in shRNA experiments. Control shRNAs are available as 20 Î¼g. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.
NOTE:Â For Western blot analysis prepare cell lysate as follows: wash cells once with PBS. Lyse cells in 300 Î¼l 1x Electrophoresis Buffer by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.
NOTE:Â 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.
NOTE:Â For a listing of many reagents required to prepare the following solutions, see Core Reagents section.
- Blotto A (for general use): 1x TBS, 5% milk, 0.05% Tween-20. Available Pre-made.
- Blotto B (for use with anti-phosphotyrosine antibodies): 1x TBS, 1% milk, 1% BSA, 0.05 Tween-20. In some cases, milk may be left out entirely, but this will result in somewhat higher backgrounds. Available pre-made. For all phospho-specific antibodies: Add 0.01% (v/v) of each Phosphatase Inhibitor Cocktail to inhibit phosphatase activity.
- Diaminobenzidine tetrahydrochloride (DAB): Dissolve 5 mg DAB in 100 ml 100 mM Tris-HCl, pH 7.6, and add 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB solution daily.
- Electrophoresis buffer (2X): 100mM 2-(N-Morpholino)- ethanesulfonic acid(MES), 10 mM Na EDTA, 15% glycerol, 1.5% SDS, 0.3% Triton X-100, 100mM TCEP-HCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Available pre-made.
- Phosphate buffered saline (1x PBS): 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, and 150 mM NaCl. Adjust pH to 7.4 with NaOH. Available pre-made in liquid and powder forms
- RIPA Lysis Buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add inhibitors at time of use from the following stock solutions. Available pre-made.
1) 10 mg/ml PMSF in isopropanol (add at 10 Âµl/ml RIPA)
2) AprotininÂ (add at 50 KIU/ml RIPA)
3) 100 mM sodium orthovanadate in frozen aliquots (add at 10 Âµl/ml RIPA)
- Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled H2O.
- Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 7.4; 150 mM NaCl. Available pre-made in liquid form.