AUMskip™ ASO Protocol
Comprehensive in vitro protocol for self-delivering exon skipping antisense oligonucleotides
Introduction
This protocol provides detailed instructions for using AUMskip™ self-delivering antisense oligonucleotides (sdASO™) in mammalian cell culture for exon skipping applications. AUMskip™ ASOs enable efficient modulation of pre-mRNA splicing without the need for transfection reagents, making them ideal for a wide range of cell types, including difficult-to-transfect cells.
Protocol Overview
AUMskip™ sdASO™ delivery is a simple, three-step process:
- Plate cells at optimal density (30-50% confluency)
- Add AUMskip™ sdASO™ directly to culture medium
- Incubate and analyze splice modulation 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
- Steric Blocking Mechanism: Modulates splicing without causing RNA degradation, enabling production of modified proteins
- Low Toxicity: Maintains cell viability without transfection reagent-associated toxicity
Materials and Reagents
Required Items
- AUMskip™ 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
- For splice analysis:
- RNA extraction reagents
- RT-PCR reagents
- Primers flanking the target exon
- Gel electrophoresis equipment (for visualization of splice variants)
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 AUMskip™ 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).
AUMskip™ sdASO™ Stock Preparation
Prepare AUMskip™ 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 AUMskip™ 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.
AUMskip™ sdASO™ Delivery to Cells
Add AUMskip™ 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 exon.
For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMskip™ 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 AUMskip™ sdASO™, or add the ASO stock directly to the media containing the cells. Mix gently.
It is highly recommended to perform a dose optimization using 2-3 working concentrations (e.g., 500 nM, 5 μM, and 10 μM) to determine the optimal concentration for your specific exon skipping application.
In some specific cases, 20 μM or higher concentrations may be required, especially for exons with strong splice sites or high expression levels.
The optimal ASO concentration may vary depending on the target exon, splice site strength, cell type, and assay timing. For most applications, 1-5 μM provides an excellent balance of efficacy and economy, but exon skipping may require higher concentrations than gene silencing applications.
Incubation and Analysis
Incubate cells with AUMskip™ sdASO™ and analyze splice modulation at appropriate time points.
Return cells to the incubator and maintain under standard culture conditions.
Analyze AUMskip™ sdASO™-treated cells after the desired time point, typically 24-72 hours post-treatment.
RNA splicing analysis can be performed using RT-PCR with primers that flank the target exon, followed by gel electrophoresis to visualize the splice variants (skipped vs. non-skipped).
For protein-level assessment, Western blot can be performed to detect the altered protein size resulting from exon skipping (typically 48-96 hours post-treatment to allow time for protein synthesis).
For splice analysis, it's critical to design primers that span the skipped exon (e.g., in adjacent exons) to clearly distinguish between splice variants. The exon-skipped product will appear as a faster-migrating band that is smaller by exactly the size of the skipped exon.
Reference Calculations
The table below provides guidelines for AUMskip™ sdASO™ amounts and cell numbers for various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMskip™ sdASO™ stock (μL)1 | 1 μL | 5 μL | 10 μL | 30 μL |
| AUMskip™ 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 AUMskip™ 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 AUMskip™ sdASO™ treatment.
Tips and Troubleshooting
Optimization Tips and Best Practices
Target Site Selection
For optimal exon skipping, AUMskip™ sdASO™ should target splice sites (3' splice acceptor or 5' splice donor) or exonic splicing enhancers within the target exon. Targeting the intron-exon junction or the first 25 nucleotides of the exon often produces the best results.
Dose Optimization
Always perform dose optimization for new targets. Exon skipping may require higher concentrations than gene silencing. Start with a concentration range (1 μM, 5 μM, and 10 μM) to determine the optimal balance between skipping efficiency and economy.
Extended Effect
The exon skipping effect can persist for several days using a single dose. For long-term experiments with rapidly dividing cells, add more AUMskip™ sdASO™ every 48-72 hours to maintain consistent exon skipping.
Controls
Include appropriate controls: untreated cells, non-targeting AUMskip™ sdASO™, and if possible, a positive control targeting a well-established skippable exon to validate your experimental system.
Troubleshooting Common Issues
Low Exon Skipping Efficiency
- Increase concentration: Try higher concentrations (up to 20 μM) as exon skipping often requires higher ASO concentrations than gene silencing.
- Test multiple target sites: Strong splice sites can be resistant to modulation. Design multiple AUMskip™ sdASO™ targeting different regions (acceptor site, donor site, and/or exonic splicing enhancers).
- Extend incubation time: Some splice modulation effects may require longer incubation (72-96 hours) for optimal results.
- Check target expression: Ensure your target gene is expressed at sufficient levels in your cell model.
Difficult Detection of Splice Variants
- Optimize PCR conditions: Adjust primer design to ensure they flank the target exon with appropriate distance for clear resolution of products.
- Use high-resolution gel: A 2-3% agarose gel or polyacrylamide gel may be needed to resolve small differences in amplicon size.
- Consider nested PCR: For low abundance transcripts, a nested PCR approach may improve detection sensitivity.
- Quantitative analysis: For subtle changes, consider using fragment analysis or qPCR with specific primers for the skipped junction.
No Protein Effect Despite RNA Skipping
- Check reading frame: Ensure the exon skipping maintains the reading frame. Out-of-frame skipping may lead to nonsense-mediated decay or truncated proteins.
- Extend time course: Protein turnover varies greatly. Allow sufficient time (72-120 hours) for existing proteins to degrade and modified proteins to accumulate.
- Verify antibody specificity: Ensure your antibody can detect the modified protein variant.
Storage and Additional Information
Storage Conditions
- AUMskip™ sdASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMskip™ 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
- AUMskip™ sdASO™ operate through steric blocking of splice sites rather than RNase H-mediated RNA degradation.
- Unlike gene silencing ASOs, AUMskip™ does not reduce total RNA levels but instead alters the ratio of splice variants.
- No pre-treatment or media change is required before adding AUMskip™ sdASO™ to cells.
- For genetic disease models, confirm that the exon skipping restores the reading frame before proceeding to functional assays.
AUMskip™ sdASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
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