- Be Consistent When Conducting Experiments
- Select Appropriate Order of Transfection
- Use Healthy Cells at the Optimal Density
- Choose the appropriate Culture Media and Culturing Conditions
- Use High Quality siRNA at the Lowest Effective Concentration
- Monitor siRNA Delivery in Every Experiment by Including Positive and Negative Control siRNAs and Untreated Samples
- Optimize Choice and Amount of Transfection Agent
- Optimize Exposure Time to Transfection Agent:siRNA Complexes
- Monitor Knockdown of Both mRNA and Protein When Possible
- Optimize Electroporation Voltage, Pulse Length, and Pulse Number in an Appropriate Buffer
All recommendations below are for siRNA, but exactly same practices can be utilized for delivery of miRNA mimics/inhibitors.
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.
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 RNAiMax), 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.
Cells should be 40–80% confluent and be used at a relatively low passage number (e.g. less than 50), that is not excessively old. 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). Too little cells or too many cells per well may also affect transfection efficiency. Try various cell densities e.g. a low, mid and high cell range based on the surface area of your desired transfection vessel.
Perform best practice tissue culture techniques. Antibiotics may not be necessary and may become increase toxicity 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. Choose the appropriate media for your cells (e.g. some cells require more glucose than others or need additional amino acids).
siRNAs should be free of reagents carried over from synthesis (e.g., ethanol, salts). 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 Untreated Samples
Negative and 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 mRNA 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 untreated samples.
The ease of transfection can vary greatly with cell line and cell type. RNAiMax transfection reagent enables efficient delivery of the small RNA cargo to a wide variety of cells, with minimal cytotoxicity. For challenging cell lines (suspension, blood, primary, neurons), Ambion scientists recommend trying at least 2 different transfection agents at different concentrations. Too little transfection reagents may limit siRNA delivery and thus gene silencing, but too much transfection agent can be toxic. Depending on the cell type, it may require different amounts of transfection reagent. If transfection proves to be unsuccessful (poor knockdown and/or viability), consider electroporation (see #10) or viral vectors for siRNA delivery.
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.
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 transfection into cells. Protein knockdown measurement is also required, in some cases, to understand the 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.
For cell lines which are not easy to transfect with the “standard” cationic lipid-based reagents,electroporation may be the only alternative. 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.
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