Short Hairpin RNA Plasmid Transfection

shRNA Plasmid Transfection

Transfection of shRNA Plasmids is a method used to introduce plasmids into host cells. Short hairpin RNA molecules (shRNA) are artificial RNA molecules with a short hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Our shRNA Plasmids typically encode three shRNA molecules that target the same gene, and an antibiotic resistance gene. Host cells are grown in transfection medium, which contains Plasmids encoding short hairpin RNA (shRNA) molecules, and an antibiotic resistance gene. Cells are exposed to antibiotics to select for the cells that express the antibiotic resistance gene. This leaves only the cells that express the plasmid, and express shRNA molecules that interfere with expression of the target gene. 

See below for a general protocol and recommendations. 


  • 6-wells tissue culture plate.
  • Deionized water.
  • Normal growth media.
  • Transfection media.
  • Fetal Bovine Serum (FBS).
  • Pipette and pipette tips.
  • Vials/tubes.
  • Antibiotic (e.g. puromycin).
  • 37°C Incubator. 

Sample and Reagent Preparation

  • Sample: In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS. Healthy cells below 70% confluence are required for successful transfection experiments. One day prior to transfection, ensure cell viability. This protocol is recommended for a single well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.
  • Transfection Working Solution: Add the shRNA Plasmid DNA solution directly to the dilute shRNA Plasmid Transfection Reagent using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature. 
  • 2x Normal Growth Medium: Prepare normal growth medium with 2 times the typical serum and antibiotics concentrations.

Note: The optimal shRNA Plasmid DNA Transfection medium  ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of deionized water as outlined below. 

  • Once the optimal shRNA Plasmid DNA Transfection medium ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/Transfection medium mix 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: Transfection medium 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.

Transfection Protocol

  1. Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step.
  2. Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.
  3. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
  4. Add the 200 μl Transfection Working Solution dropwise to the well, covering the entire layer.
  5. Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
  6. Incubate the cells 5-7 hours at 37° C in an incubator in the environmental conditions normally used to culture the cells. Longer transfection times may be desirable depending on the cell line.
  7. Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).
  8. Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.
  9. For transient transfection (recommended): aspirate media and replace with fresh 1x normal growth medium. Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the previous step.

Selection Protocol

  1. The working puromycin concentration for mammalian cell lines ranges from 1-10 μg/ml. Prior to selection, 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.
  2. 48 hours post-transfection, aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
  3. Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.