Puromycin Trending Applications: shRNA Gene Silencing Protocol

Puromycin Trending Applications: shRNA Gene Silencing Protocol

Puromycin’s longstanding application as a gene selection antibiotic has a powerful new role in genome editing utilizing the CRISPR/Cas9 system.

Here we discuss a protocol for 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: 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.

Solution B: For each transfection, dilute 1 – 6 μl of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring the final volume to 100 μl.

NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.

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 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: 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 μl 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 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 Procedure

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: The working puromycin concentration for mammalian cell lines ranges from 1-10 μg/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: 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.

NOTE: For Western blot analysis prepare cell lysate as follows: wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Buffer 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.

General Solutions

NOTE:  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 µl/ml RIPA)

2) Aprotinin  (add at 50 KIU/ml RIPA)

3) 100 mM sodium orthovanadate in frozen aliquots (add at 10 µl/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.

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