Protocol

AUMsilence sdASO protocol

In vitro protocol for self-delivering antisense oligonucleotides

AUMsilence sdASOs enable mRNA knockdown without the need for transfection reagents, in cell lines and hard-to-transfect primary cells.

Protocol overview

AUMsilence sdASO delivery is a three-step process:

01Plate cells at optimal density (50-70% confluency at treatment)
02Add AUMsilence sdASO directly to culture medium
03Incubate and analyze 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
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

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 AUMsilence sdASO.
- Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay.
02
Step 2: 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 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: AUMsilence sdASO delivery to cells
Add AUMsilence sdASO to the cells at the desired final concentration. The recommended working range is 5-20 μM, with a starting concentration of 10 μM.
- 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 the three concentrations of the range (5 μM, 10 μM and 20 μM) to determine the optimal concentration for your specific application.
Optimization tip: 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.
Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.
04
Step 4: Incubation and analysis
Incubate cells with AUMsilence sdASO and analyze knockdown at appropriate time points. Knockdown is typically observed 24 to 72 hours after treatment, depending on the cell type and the target gene. Longer time points may be required in some systems. Protein knockdown can also be observed from 24 hours after treatment. Where the target protein is long-lived, existing protein must first turn over, so a later time point may be required.
- Return cells to the incubator and maintain under standard culture conditions.
- Analyze AUMsilence sdASO-treated cells after the desired time point.
- Knockdown can be assessed by measuring target mRNA levels (qRT-PCR), protein levels (Western blot, ELISA, immunofluorescence), or functional assays relevant to your target.
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.

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 AUMsilence 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

Dose optimization

Always perform dose optimization for new cell types or targets. Start with the three concentrations of the range (5 μM, 10 μM and 20 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, 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: Move up towards 20 μM, the top of the range, for difficult targets or cell types.

Extend incubation time: Some targets may require longer incubation (72-96 hours).

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: Confirm that your target gene is expressed in your cell model under your experimental conditions.

Cell toxicity

Reduce concentration: If toxicity is observed, move down towards 5 μM, the bottom of the range.

Check cell density: Ensure cells are at optimal density (50-70% confluency) at the time of treatment.

Consider target biology: If your target is essential for cell survival, apparent toxicity may 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 3 to 5 days 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 sdASOs are shipped in lyophilized form. Upon arrival, store at -20°C.
Resuspended AUMsilence 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

AUMsilence sdASOs 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 sdASOs 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.

Note

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

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A scientist reviews the target gene, the cell type and when to read the knockdown before an order is placed.

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

AUMsilence™ sdASO™ protocol