Plasmid DNA and siRNA transfection protocol optimization

There are several experimental parameters that affect transfection efficiency and associated cell viability for siRNA and plasmid DNA transfection experiments:

• Transfection protocol (method and conditions)
• Health of cultured cells
• Purity and concentration of pDNA and/or siRNA
• Controls

Transfection protocol

Optimized transfection protocols are available for over 100 cancer cell lines and primary cell types (see Transfection Kits for Cell Lines). For in vivo delivery there are lipid based, polymer based, nanoparticle based, and PEG liposome based transfection reagents with optimized transfection protocols are available (see In Vivo Transfection Reagents), as well as tissue-targeted delivery reagents (see Kidney, Liver, Pancreas tissue targeted transfection kits).

Health of cultured cells

We do not recommend using freshly thawed cells for transfection experiments. Cells should be passaged 2-3 times and exhibit exponential growth at the time of transfection experiment. Use of antibiotics in cultured cell medium can interfere with transfection and reduce both transfection efficiency and cell viability of transfected cells. Cells and media must be free of contamination (mycoplasma, bacteria, yeast).

Cargo molecule: pDNA or siRNA

Purity and lack of degradation is very important for successful transfection experiment. Dilutions of pDNA and siRNA solutions should be done using RNase- and DNase-free water (or TE-buffer). Incomplete or low grade purification can lead to presence of such contaminants as endotoxins, proteins, lipids, carbohydrates that may induce siRNA and pDNA degradation and lack of functional performance (pDNA expression and siRNA gene silencing efficacy). Scientific Calculators can help with experimental planning, nucleic acid amount and concentration calculation, and associated conversions.

Controls

Positive and negative transfection controls are crucial for transfection experiments and associated data analysis. Commercially available siRNA and pDNA controls available from Altogen Biosystems:

• Catalog #4060 – GFP-expressing plasmid DNA (25 ug)
• Catalog #4061 – Cell Cycle Arrest siRNA (5 nmol)
• Catalog #4062 – Apoptosis Inducing siRNA (5 nmol)
• Catalog #4063 – Scrambled siRNA control, non-silencing siRNA (5 nmol)

Transfection Resource

Transfection Resources