AUMlnc™ ASO Protocol
Comprehensive in vitro protocol for self-delivering antisense oligonucleotides targeting long non-coding RNAs
Introduction
This protocol provides detailed instructions for using AUMlnc™ self-delivering antisense oligonucleotides (sdASO™) in mammalian cell culture to target long non-coding RNAs (lncRNAs). AUMlnc™ ASOs enable efficient lncRNA knockdown without the need for transfection reagents, making them ideal for studying nuclear-retained lncRNAs in a wide range of cell types.
Protocol Overview
AUMlnc™ sdASO™ delivery is a simple, three-step process:
- Plate cells at optimal density (30-50% confluency)
- Add AUMlnc™ 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.
- Nuclear Targeting: Unlike siRNAs, AUMlnc™ can effectively target nuclear-retained lncRNAs
- 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 with precise targeting of lncRNA transcripts
Materials and Reagents
Required Items
- AUMlnc™ 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 AUMlnc™ 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).
AUMlnc™ sdASO™ Stock Preparation
Prepare AUMlnc™ 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 AUMlnc™ 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.
AUMlnc™ sdASO™ Delivery to Cells
Add AUMlnc™ 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 lncRNA target.
For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMlnc™ 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 AUMlnc™ 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 lncRNA target.
For some lncRNAs with complex secondary structures or nuclear localization, higher concentrations (15-20 μM) may be required for efficient knockdown.
LncRNAs often have more complex secondary structures than mRNAs and may be less abundant. Starting with a higher concentration (5-10 μM) may yield better results for many lncRNA targets compared to typical mRNA knockdown protocols.
Incubation and Analysis
Incubate cells with AUMlnc™ sdASO™ and analyze knockdown at appropriate time points.
Return cells to the incubator and maintain under standard culture conditions.
Analyze AUMlnc™ sdASO™-treated cells after the desired time point, typically 24-72 hours post-treatment. Some nuclear lncRNAs may require 48-96 hours for optimal knockdown.
Assess knockdown by measuring target lncRNA levels using qRT-PCR, RNA-FISH, or other RNA detection methods. For functional analysis, evaluate phenotypic changes or downstream effects specific to your lncRNA of interest.
Due to nuclear retention of many lncRNAs, analysis may require special considerations. Nuclear fractionation prior to RNA isolation can help accurately assess knockdown efficiency of nuclear-retained lncRNAs. For RNA-FISH analysis, ensure your probes are specific for the lncRNA of interest.
Reference Calculations
The table below provides guidelines for AUMlnc™ sdASO™ amounts and cell numbers for various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMlnc™ sdASO™ stock (μL)1 | 1 μL | 5 μL | 10 μL | 30 μL |
| AUMlnc™ 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 AUMlnc™ sdASO™ shown yields a final concentration of 1 μM using 100 μM stock. For many lncRNA targets, higher concentrations (5-10 μM) may be optimal.
- 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 AUMlnc™ sdASO™ treatment.
Tips and Troubleshooting
Optimization Tips and Best Practices for lncRNA Targeting
Target Site Selection
LncRNAs often have complex secondary structures. For optimal knockdown, target regions with predicted high accessibility. Our AI-driven design considers RNA structure to select the most effective binding sites for your lncRNA of interest.
Nuclear lncRNA Considerations
For nuclear-retained lncRNAs, allow additional time (48-96 hours) for optimal knockdown. Nuclear lncRNAs may require higher ASO concentrations compared to cytoplasmic targets.
Functional Validation
Since lncRNAs can function through various mechanisms, confirm the functional consequences of knockdown beyond just measuring RNA levels. This may include evaluating expression of genes regulated by the lncRNA, chromatin modifications, or specific cellular phenotypes.
Controls
Include appropriate controls: untreated cells, scrambled/non-targeting AUMlnc™ sdASO™, and positive control ASOs. For lncRNAs with adjacent or overlapping coding genes, carefully monitor effects on neighboring genes to confirm specificity.
Troubleshooting Common Issues with lncRNA Targeting
Low Knockdown Efficiency
- Increase concentration: LncRNAs often require higher concentrations. Try 10-20 μM for challenging lncRNA targets.
- Extend incubation time: Nuclear lncRNAs may require 48-96 hours for optimal knockdown due to nuclear accessibility and turnover rates.
- Target accessibility: LncRNAs can have extensive secondary structures. Consider ordering alternative AUMlnc™ sdASO™ targeting different regions of the same lncRNA.
- Verify detection method: Ensure your qRT-PCR primers or RNA-FISH probes target the same region of the lncRNA as your ASO and consider nuclear fractionation for nuclear-retained lncRNAs.
Specificity Concerns
- Check for overlapping genes: Many lncRNAs are transcribed from genomic regions that overlap or are adjacent to coding genes. Monitor expression of neighboring genes to confirm specificity.
- Examine antisense transcripts: If your lncRNA has an antisense transcript, ensure your knockdown and detection methods are strand-specific.
- Use multiple ASOs: Target different regions of the same lncRNA to confirm that observed phenotypes are due to lncRNA knockdown rather than off-target effects.
No Phenotypic Effect Despite Confirmed Knockdown
- Functional redundancy: Some lncRNAs have functional redundancy with other transcripts. Consider combinatorial targeting approaches.
- Cell-type specificity: LncRNAs often function in specific cellular contexts. Ensure your experimental system is appropriate for the lncRNA's known biological function.
- Timing considerations: Some lncRNA-mediated effects may require longer observation periods or specific cellular stimuli to become apparent.
- Examine alternative readouts: The lncRNA's function may be subtle or context-dependent. Consider RNA-seq or other global analyses to detect broader impacts on gene expression or cellular pathways.
Storage and Additional Information
Storage Conditions
- AUMlnc™ sdASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMlnc™ 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 for lncRNA Research
- AUMlnc™ sdASO™ efficiently enter both the cytoplasm and nucleus, making them ideal for targeting nuclear-retained lncRNAs that are inaccessible to traditional RNAi approaches.
- The nuclear penetration capability of AUMlnc™ sdASO™ can be particularly valuable for studying lncRNAs involved in chromatin regulation, nuclear architecture, or transcriptional control.
- For lncRNAs with multiple isoforms, consider designing ASOs that target shared exons or specific variants depending on your research question.
- When targeting lncRNAs with regulatory functions, consider evaluating downstream gene expression changes as a measure of functional knockdown.
AUMlnc™ sdASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
Ready to Advance Your lncRNA Research?
Our scientific team is available to help you design optimal AUMlnc™ sdASO™ for your specific lncRNA target and application. Contact us for personalized support or to request a quote.
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