shRNA Transient Transfection Protocol
This sample protocol is for experiments performed in 6-well plates. If performing experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. OriGene recommends using TurboFectin 8.0 (Cat # TF81001) for all transfections, as it consistently results in high transfection efficiency.
Step 1, Preparation of cells:
- Approximately 18-24 hours prior to transfection, plate the appropriate cells (e.g. HEK293 for human, NIH3T3 for mouse or OLN-93 for rat shRNA validation) at 3 X 105 in 2 ml into the well of a 6-well plate. Grow the cells overnight in a 5% CO2 incubator to achieve 50% confluence.
Step 2, Preparation of the Turbofectin 8.0/DNA complexes (prepare immediately prior to transfection):
- Add 50 uL of dH2O into each of the tubes containing shRNA expression plasmids. Vortex the tubes briefly to resuspend the DNA. The concentration of this solution is 100 ng/uL.
- In a small sterile tube, combine the following reagents in the prescribed order. The order of reagent addition is important to achieve the optimal results.
- Dilute 1 μg of DNA in 250 uL of Opti-MEM I (Gibco 51985). Vortex gently.
- Add 3 uL of Turbofectin 8.0 to the diluted DNA (not the reverse order) and Pipette gently to mix completely.
- Incubate for 15 minutes at room temperature.
cDNA expression plasmid for the target gene 0.01 ug to 1.0 ug (optional, available at OriGene)
Note: Add TurboFection 8.0 (or equivalent) directly into the serum-free media. DO NOT let the transfection reagent touch any plastic other than the pipette tip. For Dual-gene knockdown experiment, add 500ng of each shRNA expression plasmids (both pGFP-V-RS vector and pRFP-C-RS vector together) with 500 ng each of target cDNAs.
Step 3, Transfection:
- Add the mixture prepared in Step 2 dropwise to the cells. Gently rock the plate back-and-forth and from side-to-side to distribute the complex evenly.
- Incubate the cells in a 5% CO2 incubator for 48 hrs before harvesting for RNA analysis and 72 hrs before harvesting for protein analysis.