AUMsaver™ ASO Protocol
Comprehensive in vitro protocol for transfection-optimized antisense oligonucleotides
Introduction
This protocol provides detailed instructions for using AUMsaver™ transfection-optimized antisense oligonucleotides (toASO™) in mammalian cell culture. AUMsaver™ ASOs deliver efficient mRNA knockdown at a budget-friendly price point, making them ideal for high-throughput screening and routine gene silencing in standard cell lines.
Protocol Overview
AUMsaver™ toASO™ delivery requires standard transfection methods. This protocol covers several approaches:
- Lipid-based transfection (most common method)
- Electroporation (for difficult-to-transfect cells)
- Calcium phosphate transfection (economical option)
- Polymer-based transfection
This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.
- Cost-Effective: Budget-friendly option for high-throughput studies and large-scale experiments
- High Specificity: Minimal off-target effects compared to siRNA approaches
- Flexible Delivery: Compatible with all standard transfection methods
- Same AUMsilence Platform: Maintains the core advantages of our AUMsilence technology - high potency, specificity, and stability
Materials and Reagents
Required Items
- AUMsaver™ toASO™ (lyophilized or stock solution)
- Appropriate cell culture medium
- Serum-free medium (for lipid-based transfection)
- Culture plates or vessels
- Mammalian cells of interest
- Sterile nuclease-free water or buffer (for ASO resuspension)
- Microcentrifuge tubes (for aliquoting ASO stock)
- Transfection reagent of choice:
- Lipid-based transfection reagent
- Electroporation system (if using electroporation)
- Calcium phosphate transfection reagents (if using this method)
- Polymer-based transfection reagent
- 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 transfection at the appropriate density (typically 60-80% confluency at the time of transfection). Allow cells to adhere overnight.
For suspension cells: Prepare cells at the appropriate density shortly before transfection with AUMsaver™ toASO™.
Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay. As a general guideline, aim for 60-80% confluency at the time of transfection for adherent cells.
For lipid-based transfection in 24-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).
AUMsaver™ toASO™ Stock Preparation
Prepare AUMsaver™ toASO™ stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.
Resuspend lyophilized AUMsaver™ toASO™ 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.
Transfection Method Selection
AUMsaver™ toASO™ can be delivered using various transfection methods. Choose the most appropriate method for your cell type and experimental needs.
Lipid-based transfection: Most common and versatile method, suitable for a wide range of cell types.
Electroporation: Effective for difficult-to-transfect cells, including primary cells and some immune cells.
Calcium phosphate transfection: Economical option suitable for some adherent cell lines.
Polymer-based transfection: Alternative method that works well for certain cell types.
If you are unsure which method to use, lipid-based transfection is recommended as a starting point for most cell types. For difficult-to-transfect cells, electroporation often provides the best results.
Lipid-Based Transfection Protocol
This protocol describes a general lipid-based transfection method for AUMsaver™ toASO™. Always refer to the specific instructions provided by the manufacturer of your transfection reagent.
Dilute AUMsaver™ toASO™: In a sterile tube, dilute AUMsaver™ toASO™ in serum-free medium to achieve the desired concentration. For a 24-well plate, typically use 50 μL of serum-free medium and 1-2 μL of 100 μM AUMsaver™ toASO™ stock (final concentration in well: 20-50 nM).
Dilute transfection reagent: In a separate sterile tube, dilute the transfection reagent in serum-free medium according to the manufacturer's recommendations. For a 24-well plate, typically use 50 μL of serum-free medium and 1-3 μL of transfection reagent.
Combine and incubate: Add the diluted transfection reagent to the diluted AUMsaver™ toASO™ (not the reverse). Mix gently by pipetting up and down or tapping the tube. Incubate at room temperature for 10-20 minutes to allow complexes to form.
Add complexes to cells: Add the transfection complex solution dropwise to the cells. Gently rock the plate to ensure even distribution.
Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing knockdown efficiency.
Some transfection reagents require a media change 4-6 hours post-transfection to minimize toxicity. Refer to the manufacturer's guidelines for your specific transfection reagent.
Electroporation Protocol
Electroporation is an effective method for delivering AUMsaver™ toASO™ to difficult-to-transfect cells. The exact parameters will depend on your electroporation system and cell type.
Prepare cells: Harvest cells in the growth phase and wash with PBS. Resuspend cells in the appropriate electroporation buffer at a density recommended for your electroporation system (typically 1-5 × 106 cells/mL).
Add AUMsaver™ toASO™: Add AUMsaver™ toASO™ to the cell suspension to achieve a final concentration of 1-2 μM. Mix gently.
Electroporate: Transfer the cell/ASO mixture to electroporation cuvettes. Perform electroporation using parameters optimized for your cell type (commonly used settings: 200-450V, 1-3 ms pulse length).
Recovery: Immediately after electroporation, add pre-warmed complete medium to the cells. Transfer the cells to culture plates or flasks.
Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing knockdown efficiency.
Electroporation parameters vary significantly between cell types and electroporation systems. Always optimize the electroporation conditions for your specific cells and equipment.
Calcium Phosphate Transfection Protocol
Calcium phosphate transfection is an economical method suitable for some adherent cell lines.
Prepare Solution A: In a sterile tube, mix AUMsaver™ toASO™ (1-2 μL of 100 μM stock for a 24-well plate) with 125 mM CaCl2 solution to a total volume of 25 μL.
Prepare Solution B: In a separate tube, add 25 μL of 2X HBS buffer (HEPES-buffered saline, pH 7.05-7.12).
Form precipitate: Add Solution A dropwise to Solution B while gently vortexing or bubbling air through Solution B using a pipette. Incubate at room temperature for 20-30 minutes to allow precipitate to form.
Add to cells: Add the transfection mixture dropwise to cells in complete medium. Gently rock the plate to ensure even distribution.
Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing knockdown efficiency.
The pH of the 2X HBS is critical for efficient transfection. Small variations in pH can significantly affect transfection efficiency.
Polymer-Based Transfection Protocol
Polymer-based transfection is an alternative method that works well for certain cell types.
Dilute AUMsaver™ toASO™: In a sterile tube, dilute AUMsaver™ toASO™ in the buffer recommended by the polymer transfection reagent manufacturer. For a 24-well plate, typically use 50 μL of buffer and 1-2 μL of 100 μM AUMsaver™ toASO™ stock.
Dilute polymer reagent: In a separate sterile tube, dilute the polymer transfection reagent in the appropriate buffer according to the manufacturer's recommendations.
Combine and incubate: Add the diluted polymer reagent to the diluted AUMsaver™ toASO™. Mix gently and incubate at room temperature for the time specified by the manufacturer (typically 10-15 minutes).
Add complexes to cells: Add the transfection complex solution dropwise to the cells. Gently rock the plate to ensure even distribution.
Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing knockdown efficiency.
Always follow the specific instructions provided by the manufacturer of your polymer-based transfection reagent, as protocols can vary significantly between products.
Analysis of Knockdown Efficiency
Evaluate the knockdown efficiency of AUMsaver™ toASO™ at appropriate time points post-transfection.
Timing: Assess knockdown at 24-72 hours post-transfection, depending on your target gene's mRNA and protein half-life.
mRNA analysis: Use quantitative RT-PCR to measure target mRNA levels relative to a housekeeping gene.
Protein analysis: Use Western blot, ELISA, or immunofluorescence to assess target protein levels.
Functional assays: Perform relevant functional assays to evaluate the phenotypic effects of target gene knockdown.
Always include appropriate controls in your experiments: untreated cells, cells treated with transfection reagent only, scrambled/non-targeting AUMsaver™ toASO™, and positive control ASOs targeting housekeeping genes with known knockdown efficiency.
Reference Calculations
The table below provides guidelines for AUMsaver™ toASO™ amounts and transfection reagent volumes for lipid-based transfection in various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMsaver™ toASO™ (100 μM stock) | 0.2-0.4 μL | 1-2 μL | 2-4 μL | 5-10 μL |
| Serum-free medium for ASO dilution | 10 μL | 50 μL | 100 μL | 250 μL |
| Transfection reagent1 | 0.2-0.6 μL | 1-3 μL | 2-6 μL | 5-15 μL |
| Serum-free medium for reagent dilution | 10 μL | 50 μL | 100 μL | 250 μL |
| Complete medium in well | 100 μL | 500 μL | 1000 μL | 2000 μL |
| Cell number (per well)2 | 1 × 104 | 0.5 × 105 | 1 × 105 | 2.5 × 105 |
- The amount of transfection reagent can vary significantly depending on the specific product and cell type. Always refer to the manufacturer's recommendations.
- The optimal seeding cell density will vary with the cell type, cell size, growth characteristics, and the endpoint of the assay. The values shown are for HeLa cells at 70% confluency at the time of transfection.
Tips and Troubleshooting
Optimization Tips and Best Practices
Optimize Transfection Conditions
For each new cell type or transfection reagent, optimize the ASO:reagent ratio and total concentration. Test 2-3 different ratios to find the optimal balance between knockdown efficiency and cell viability.
Cell Density Matters
Cell confluency at the time of transfection significantly impacts efficiency. For lipid-based transfection, 60-80% confluency is typically optimal. Too low or too high cell density can reduce transfection efficiency.
Serum Considerations
Some transfection reagents are inhibited by serum. If recommended by the manufacturer, perform the transfection in serum-free medium and replace with complete medium 4-6 hours post-transfection.
Antibiotics
For sensitive cells, consider removing antibiotics from the medium during transfection, as the combination of transfection reagents and antibiotics can increase toxicity.
Troubleshooting Common Issues
Low Transfection Efficiency
- Optimize ASO:transfection reagent ratio: Test different ratios to find the optimal balance.
- Check cell density: Ensure cells are at the appropriate confluency (typically 60-80%) at the time of transfection.
- Verify transfection reagent quality: Some reagents lose potency over time or with multiple freeze-thaw cycles.
- Try a different transfection method: If lipid-based transfection yields poor results, consider electroporation or polymer-based transfection.
- Increase ASO concentration: For difficult targets, try increasing the ASO concentration up to 100-200 nM final concentration.
High Cell Toxicity
- Reduce transfection reagent amount: Excessive transfection reagent can be toxic to cells.
- Change medium post-transfection: Replace the transfection medium with fresh complete medium 4-6 hours after transfection.
- Remove antibiotics: For sensitive cells, antibiotics in the medium can increase transfection-related toxicity.
- Reduce ASO concentration: If toxicity persists, try decreasing the ASO concentration while maintaining the optimal ASO:reagent ratio.
- Consider target biology: If your target is essential for cell survival, apparent toxicity may actually be a consequence of successful knockdown.
Poor Knockdown Despite Good Transfection
- Verify ASO design: The ASO might target a region of the mRNA that is structurally inaccessible. Consider ordering a different AUMsaver™ toASO™ 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.
- Extend incubation time: Some proteins have long half-lives and may require 72-96 hours post-transfection for significant reduction at the protein level.
- Verify qPCR primers: Ensure your qPCR primers target a region different from the ASO binding site and that they detect all relevant transcript variants.
Storage and Additional Information
Storage Conditions
- AUMsaver™ toASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMsaver™ toASO™ 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
- AUMsaver™ toASO™ are optimized for standard transfection methods but do not self-deliver like our sdASO™ products.
- Always follow the specific instructions provided by the manufacturer of your transfection reagent, as protocols can vary significantly between products.
- For phenotypic assays, the timing should be optimized based on both the knockdown kinetics and the stability of the target protein.
- AUMsaver™ toASO™ can be used for sequential or co-transfection to knockdown multiple genes simultaneously.
AUMsaver™ toASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
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Related Resources
AUMsilence™ Protocol
Detailed protocol for mRNA knockdown using our self-delivering AUMsilence™ sdASO™.
View ProtocolTransfection Optimization Guide
Comprehensive guide for optimizing transfection conditions for different cell types.
View Guide