AUMsilence™ ASO Protocol
Comprehensive in vitro protocol for self-delivering antisense oligonucleotides
Introduction
This protocol provides detailed instructions for using AUMsilence™ self-delivering antisense oligonucleotides (sdASO™) in mammalian cell culture. AUMsilence™ ASOs enable efficient mRNA knockdown without the need for transfection reagents, making them ideal for a wide range of cell types, including difficult-to-transfect cells.
Protocol Overview
AUMsilence™ sdASO™ delivery is a simple, three-step process:
- Plate cells at optimal density (30-50% confluency)
- Add AUMsilence™ sdASO™ directly to culture medium
- Incubate and analyze results (typically 24-72 hours)
This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.
- No Transfection Required: Simply add to media - no lipofection, electroporation, or viral vectors needed
- Universal Cell Compatibility: Works in difficult-to-transfect cell types including primary cells, neurons, and immune cells
- High Specificity: Minimal off-target effects compared to siRNA approaches
- Low Toxicity: Maintains cell viability without transfection reagent-associated toxicity
Materials and Reagents
Required Items
- AUMsilence™ sdASO™ (lyophilized or stock solution)
- Appropriate cell culture medium
- Culture plates or vessels
- Mammalian cells of interest
- Sterile nuclease-free water or buffer (for ASO resuspension)
- Microcentrifuge tubes (for aliquoting ASO stock)
- Standard cell culture equipment:
- Sterile pipettes and tips
- Cell culture hood
- Humidified cell culture incubator
- Centrifuge
Detailed Protocol
Cell Preparation
Plate cells in their optimum growth medium at appropriate density for your cell type.
For adherent cells: Plate cells the day before treatment at 30-50% confluency (or at densities optimized for your specific cell type and assay endpoint). Allow cells to adhere overnight.
For suspension cells: Prepare cells at appropriate density shortly before treatment with AUMsilence™ sdASO™.
Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay. As a general guideline, aim for 30-50% confluency at the time of ASO treatment.
When using 96-well plates, approximately 0.5 × 105 cells per well is recommended for cells similar to HeLa in size. Scale accordingly for different plate formats (see reference table below).
AUMsilence™ sdASO™ Stock Preparation
Prepare AUMsilence™ sdASO™ stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.
Resuspend lyophilized AUMsilence™ sdASO™ using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 100 μM).
Pipette the solution up and down 3-5 times while avoiding the introduction of bubbles.
Let the vial sit at room temperature for 5-10 minutes to ensure complete resuspension.
Centrifuge for 30-45 seconds to collect the solution at the bottom of the tube.
Prepare several aliquots of the stock solution to avoid multiple freeze-thaw cycles.
To avoid degradation, minimize freeze-thaw cycles of your ASO stock. It is strongly recommended to make single-use aliquots of your stock solution and store them at -20°C.
AUMsilence™ sdASO™ Delivery to Cells
Add AUMsilence™ sdASO™ to the cells at the desired final concentration. The working concentration can vary from 500 nM to 10 μM depending on the cell type and target gene.
For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMsilence™ sdASO™, or add the ASO stock directly to the media overlaying the cells. Mix gently.
For suspension cells: Either pellet the cells by low-speed centrifugation and gently resuspend the cell pellet in media containing AUMsilence™ sdASO™, or add the ASO stock directly to the media containing the cells. Mix gently.
It is highly recommended to perform a dose response using 2-3 working concentrations (e.g., 500 nM, 5 μM, and 10 μM) to determine the optimal concentration for your specific application.
In some specific cases, 20 μM or higher concentrations may be required.
The optimal ASO concentration may vary depending on the target gene, cell type, and assay timing. For most applications, 1-5 μM provides an excellent balance of efficacy and economy.
Incubation and Analysis
Incubate cells with AUMsilence™ sdASO™ and analyze knockdown at appropriate time points.
Return cells to the incubator and maintain under standard culture conditions.
Analyze AUMsilence™ sdASO™-treated cells after the desired time point, typically 24-72 hours post-treatment.
Knockdown can be assessed by measuring target mRNA levels (qRT-PCR), protein levels (Western blot, ELISA, immunofluorescence), or functional assays relevant to your target.
Uptake of fluorescently labeled AUMsilence™ sdASO™ can be observed as early as 4-8 hours post-treatment, but full knockdown assessment is best performed at 24-96 hours post-treatment, depending on the target's mRNA and protein half-life.
Reference Calculations
The table below provides guidelines for AUMsilence™ sdASO™ amounts and cell numbers for various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMsilence™ sdASO™ stock (μL)1 | 1 μL | 5 μL | 10 μL | 30 μL |
| AUMsilence™ sdASO™ used (moles) | 100 pmole | 500 pmole | 1 nmole | 3 nmole |
| Cell culture media (μL) | 100 μL | 500 μL | 1000 μL | 3000 μL |
| Cell number (per well)2 | 0.5 × 105 | 2.5 × 105 | 0.5 × 106 | 1 × 106 |
- The amount of AUMsilence™ sdASO™ shown yields a final concentration of 1 μM using 100 μM stock.
- The optimal seeding cell density will vary with the cell type, cell size, growth characteristics, and the endpoint of the assay. For this table, HeLa cells at 50% confluency were used at the time of AUMsilence™ sdASO™ treatment.
Tips and Troubleshooting
Optimization Tips and Best Practices
Dose Optimization
Always perform dose optimization for new cell types or targets. Start with a concentration range (500 nM, 5 μM, and 10 μM) to determine the optimal balance between knockdown efficiency and economy.
Extended Knockdown
For long-term experiments, knockdown can be maintained for several days (and weeks in some cases) using a single dose. For very fast-growing cells where the knockdown effect may diminish after a few days, simply add more AUMsilence™ sdASO™ to the cell culture to maintain knockdown.
Fluorescent Monitoring
AUMsilence™ sdASO™ can be ordered with fluorescent labels to monitor cellular uptake in real-time using fluorescence microscopy or flow cytometry.
Controls
Include appropriate controls in your experiments: untreated cells, scrambled/non-targeting AUMsilence™ sdASO™, and positive control ASOs targeting housekeeping genes with known knockdown efficiency.
Troubleshooting Common Issues
Low Knockdown Efficiency
- Increase concentration: Try higher concentrations (up to 20 μM) for difficult targets or cell types.
- Extend incubation time: Some targets may require longer incubation (72-96 hours) for optimal knockdown.
- Verify target accessibility: The target sequence might have structural constraints. Consider ordering a different AUMsilence™ sdASO™ targeting a different region of the same gene.
- Check target expression: Ensure your target gene is expressed in your cell model under your experimental conditions.
Cell Toxicity
- Reduce concentration: If toxicity is observed, try lower concentrations (500 nM - 1 μM).
- Check cell density: Ensure cells are at optimal density (30-50% confluency) at the time of treatment.
- Consider target biology: If your target is essential for cell survival, apparent toxicity may actually be a consequence of successful knockdown.
Rapid Loss of Knockdown
- For rapidly dividing cells: The ASO concentration may be diluted by cell division. Add additional ASO every 48-72 hours to maintain knockdown.
- Check target mRNA turnover: Genes with high transcription rates may require higher ASO concentrations or repeated treatments.
Storage and Additional Information
Storage Conditions
- AUMsilence™ sdASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMsilence™ sdASO™ should be stored in aliquots at -20°C to avoid multiple freeze-thaw cycles.
- For short-term storage (up to 1 week), resuspended ASOs can be kept at 4°C.
Additional Notes
- AUMsilence™ sdASO™ are compatible with standard cell culture media, including those containing serum.
- No pre-treatment or media change is required before adding AUMsilence™ sdASO™ to cells.
- AUMsilence™ sdASO™ are not affected by antibiotics in the culture medium.
- For phenotypic assays, the timing should be optimized based on both the knockdown kinetics and the stability of the target protein.
AUMsilence™ sdASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
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