siRNA can be produced by chemical synthesis, shRNA plasmid and shRNA viral vector transcription. For siRNA transfection gene silencing experiments, appropriate positive and negative controls must be setup. The negative control normally include a scrambled/mismatched siRNA oligo containing the scrambled/mismatched sequence of the siRNA. The positive control may include a publically validated siRNA oligo for an unrelated gene. As other RNA techniques, precautions should be taken to avoid RNase contamination. To obtain the highest siRNA transfection efficiency (70% and up) while minimizing the toxicity, user needs to optimize the siRNA transfection using siRNA transfection reagent and subconfluent (50-70% confluency) cells. Off-target effect of siRNA should be avoided by sequence analysis using BLAST tool. The siRNA sequence should only be targeted on the gene of interests.

General procedure of siRNA transfection:

1. Plate cells in 6 or 24 well plate in culture media w/o antibiotics.

2. Grow cells until 50-70% confluent (18-24 hours).

3. Prepare the siRNA transfection solutions and incubate in room temperature for 15-45 min.

4. Wash cells with antibiotic and serum free medium.

5. Dilute and mix gently the siRNA transfection solution with serum/antibiotics free medium. Add in the wells to cover washed cells.

6. Incubate cells in CO2 incubator for 5-7 hours.

7. Replace siRNA transfection medium with fresh culture medium.

8. Culture 24-72 hours (for short-term transfection) and harvest cells for downstream assay. Western blot at protein level and RT-PCR at mRNA level can be used to analyze the siRNA knock down efficiency.

siRNA Transfection Protocols

1. GeneTran reagent DNA and siRNA transfection reagent protocol.
2. DNA and siRNA transfection protocols.

Some Cationic lipid-based siRNA transfection protocols:

1. Protocol for siRNA transfection into mammalian cells using Invitrogen Lipofectamine RNAiMax transfection reagent.
2. Protocol for siRNA transfection into mammalian cells using Invitrogen Lipofectamine 2000 transfection reagent.
3. Plasmid and siRNA cotransfection protocol using Lipofectamine 2000.
4. siRNA Transfection Protocol for TransPass R1 from NEB.
5. siRNA Transfection Protocol for DharmaFECT from Thermo Scientific.
6. siIMPORTER siRNA Transfection Protocol.

Technical articles:

1. Optimized Basic Conditions Are Essential for Successful siRNA Transfection into Primary Endothelial Cells. Andrea Nolte, Claudia Raabe, Tobias Walker, Perikles Simon, Gerhard Ziemer, Hans Peter Wendel. Oligonucleotides. June 2009, 19(2): 141-150. doi:10.1089/oli.2009.0182.
2. siRNA transfection wiki article from OpenWetWare.