Protocol

AUMsplice sdASO protocol

In vitro protocol for self-delivering exon skipping antisense oligonucleotides

AUMskip sdASO is the exon-skipping application of AUMsplice sdASO. AUMsplice sdASOs enable modulation of pre-mRNA splicing without the need for transfection reagents, in cell lines and hard-to-transfect primary cells.

Protocol overview

AUMsplice sdASO delivery is a three-step process:

01Plate cells at optimal density (50-70% confluency at treatment)
02Add AUMsplice sdASO directly to culture medium
03Incubate and analyze splice modulation results (typically 24-72 hours after treatment)

This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.

Key advantages

No transfection required: Add to media (no lipofection, electroporation, or viral vectors needed)
Cell compatibility: Works in difficult-to-transfect cell types including primary cells, neurons, and immune cells
Steric blocking mechanism: Modulates splicing without recruiting RNase H, enabling production of modified proteins
Low toxicity: Maintains cell viability without transfection reagent-associated toxicity

Materials and reagents

Required items

AUMsplice 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

01
Step 1: Cell preparation
Plate cells in their optimum growth medium at a density appropriate for the cell type. A confluency of 50-70% at the time of treatment is typical.
- For adherent cells: Plate cells the day before treatment, at a seeding density that brings them to that confluency by the time of treatment (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 AUMsplice sdASO.
- Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay.
02
Step 2: AUMsplice sdASO stock preparation
Prepare AUMsplice 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 AUMsplice sdASO using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 1 mM).
- 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.
Important: 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.
Cells seeded, here in a 24-well plate; the oligonucleotide waits in solution.
03
Step 3: AUMsplice sdASO delivery to cells
Add AUMsplice sdASO to the cells at the desired final concentration. The recommended working range is 5-20 μM, with a starting concentration of 10 μM. The optimal concentration varies with the target gene, the RNA class (messenger RNA, microRNA or long non-coding RNA) and the cell type, and should be determined by titration for each system.
- For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMsplice 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 AUMsplice 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 the three concentrations of the range (5 μM, 10 μM and 20 μM) to determine the optimal concentration for your specific exon skipping application.
Optimization tip: An exon may need more than a message does.
Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.
04
Step 4: Incubation and analysis
Incubate cells with AUMsplice sdASO and analyze splice modulation at appropriate time points.
- Return cells to the incubator and maintain under standard culture conditions.
- Analyze AUMsplice 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).
Note: For splice analysis, it is 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.
Return the plate to the humidified incubator under standard culture conditions, typically 24-72 hours.

Reference calculations

Making the stock

Reconstitute the vial in nuclease-free water to 1 mM (1 μL per nmol, so 25 μL for a 25 nmol vial). For a 100 μM stock, add 10 μL per nmol.

Vial	For 1 mM	For 100 μM
10 nmol	10 μL	100 μL
25 nmol	25 μL	250 μL
50 nmol	50 μL	500 μL
100 nmol	100 μL	1 mL

Adding the stock to the well

Add the 1 mM stock to the well at 1:200 for 5 μM, 1:100 for 10 μM and 1:50 for 20 μM. At 10 μM that is 1 μL per 100 μL of medium.

Plate	Medium in the well	5 μM	10 μM	20 μM
96-well	100 μL	0.5 μL	1 μL	2 μL
48-well	250 μL	1.25 μL	2.5 μL	5 μL
24-well	500 μL	2.5 μL	5 μL	10 μL
12-well	1 mL	5 μL	10 μL	20 μL
6-well	2 mL	10 μL	20 μL	40 μL

Treatments per vial

At 10 μM, a 25 nmol vial treats 25 wells of a 96-well plate, 5 of a 24-well plate or 2 of a 12-well plate. Counts are rounded down to whole wells. AUMsplice sdASO is supplied by quotation in these vials.

Plate	Medium in the well	5 μM	10 μM	20 μM
96-well	100 μL	50	25	12
48-well	250 μL	20	10	5
24-well	500 μL	10	5	2
12-well	1 mL	5	2	1
6-well	2 mL	2	1	0

Cell density at treatment

Add AUMsplice sdASO when adherent cells are at 50-70% confluency. Seed suspension cells and primary cells at the density their cell type guide gives.

Tips and troubleshooting

Optimization tips and best practices

Target site selection

AUMsplice 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 need more than gene silencing does. Start with the three concentrations of the range (5 μM, 10 μM and 20 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 AUMsplice sdASO every 3 to 5 days to maintain consistent exon skipping. Non-dividing cells may not need a further dose for 10 to 14 days.

Controls

Include appropriate controls: untreated cells, non-targeting AUMsplice 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: Move up towards 20 μM, the top of the range, as exon skipping often requires higher ASO concentrations than gene silencing.

Test multiple target sites: Strong splice sites can be resistant to modulation. Consider ordering multiple AUMsplice sdASOs 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).

Check target expression: Confirm that 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

AUMsplice sdASOs are shipped in lyophilized form. Upon arrival, store at -20°C.
Resuspended AUMsplice sdASOs 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

AUMsplice sdASOs operate through steric blocking of splice sites rather than RNase H-mediated RNA degradation.
Unlike gene silencing ASOs, AUMsplice sdASO alters the ratio of splice variants rather than cleaving the target. Total RNA may still fall where the new variant carries a premature termination codon, since the cell degrades such transcripts. Read the variant ratio rather than the total.
No pre-treatment or media change is required before adding AUMsplice sdASO to cells.
For genetic disease models, confirm that the exon skipping restores the reading frame before proceeding to functional assays.

Note

AUMsplice sdASOs are for research use only. Not for use in diagnostic or therapeutic procedures.

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A scientist reviews the target exon, the splice site to block and how the variants will be read before an order is placed.

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For research use only. Not for use in diagnostic or therapeutic procedures.

AUMsplice™ sdASO™ protocol