Top Ten Ways to Optimize siRNA Delivery in Cultured Cells
. Use healthy cells at the optimal density
Cells should be 20–80% confluent and be used at a passage number that is well characterized. Over time, cell cultures often diverge in phenotype and growth characteristics, and thus in expression profile, compared to cells from earlier passages. Many cells will also undergo expression profile changes when they are stressed by culture conditions (e.g., frequent temperature and pH changes, inadequate nutrition from medium depletion in overgrown cultures, instability from subculturing at too low a cell density, extended exposure to trypsin, exposure to shear forces from vigorous pipetting or centrifugation, and mycoplasma contamination
. Use high quality siRNA at the lowest effective concentration.
siRNAs should be free of reagents carried over from synthesis (e.g., ethanol, salts, and proteins). Double-stranded RNA contaminants longer than 30 bp can alter gene expression by activating the nonspecific interferon response, resulting in cytotoxicity. Ambion’s standard purity siRNAs are free of these contaminants and are ideal for cell culture experiments. During delivery optimization experiments, determine the lowest effective concentration of siRNAs to use with your delivery method and cell type(s); using more siRNA than needed increases the chance of nonspecific (off-target) effects
Monitor siRNA delivery in every experiment by including positive and negative control siRNAs and nontreated samples
Positive control siRNAs should be included in every experiment to help monitor siRNA delivery efficiency (i.e., compare target gene expression levels in cells treated with the positive control siRNA with that of cells treated with the negative control siRNA). For most validated control siRNAs and cell systems, you should see ≥70% knockdown of the control siRNA target. In addition, cytotoxicity from the siRNA delivery method should be monitored by comparing viable cell numbers in cultures that were treated with the negative control siRNA to that of nontreated samples
. Monitor knockdown of both mRNA and protein levels when possible.
Measuring mRNA knockdown is the most direct way to monitor the effects of siRNA treatment. Target mRNA levels are typically quantitated 24–48 hours after siRNA delivery. Protein knockdown is also required to see biological effects of siRNA treatment and is typically assessed 48–72 hours after siRNA delivery. Decreases in protein expression are affected by the rate of mRNA and protein turnover; the optimal time to observe maximal protein knockdown may vary widely for different targets
. Optimize culture media.
Antibiotics may become toxic if taken up during siRNA delivery. Also, some siRNA transfection agents require that transfection be carried out in serum-free or reduced-serum medium, so be sure to note the manufacturer’s recommendations
. Be consistent when conducting experiments.
As conditions are optimized, care should be taken to be consistent in the order, timing, and manner of conducting each step in the protocol. Your results will be more reproducible and reliable if there is less variation across experiments
. Optimize choice and amount of transfection agent.
The ease of transfection can vary greatly with cell line and cell type. Ambion scientists recommend trying at least 2–3 different transfection agents at different concentrations. Too little transfection agent may limit siRNA delivery and thus gene silencing, but too much transfection agent can be toxic. If transfection proves to be unsuccessful (poor knockdown and/or viability), consider electroporation (see #10) or viral vectors for siRNA delivery
. Select appropriate order of transfection.
In standard transfection protocols, cells are plated ~24 hours prior to transfection. Reverse transfection allows cells to be plated and transfected simultaneously, saving an entire day in the procedure. Ambion scientists have found that when using efficient, reverse transfection protocols (e.g., as with siPORT™ NeoFX™ Transfection Agent), some cell lines are more effectively transfected, siRNA concentrations can often be decreased (which may reduce the chance of off-target effects), and a broader range of cell concentrations can be used successfully.
. Optimize exposure time to transfection agent:siRNA complexes.
Transfection efficiency is influenced by the amount of transfection agent:siRNA complexes. If cytotoxicity and target gene knockdown are both high, you may be able to reduce the levels of cytotoxicity while maintaining strong gene silencing by replacing media containing transfection agent:siRNA complexes with normal growth media 8–24 hours after transfection
Optimize electroporation voltage, pulse length, and pulse number in an appropriate buffer.
The voltage, pulse length, and pulse number are all important factors in inducing transient cell membrane permeability by electroporation with square wave type pulses. Similar to transfection procedures, there is a balance between target gene knockdown and cell mortality as electroporation conditions are varied
For more information on siRNA delivery, check out the siRNA Delivery Resource at www.ambion.com/RNAi/delivery.