Protocol

AUMlnc sdASO protocol

In vitro protocol for self-delivering antisense oligonucleotides targeting long non-coding RNAs

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

Protocol overview

AUMlnc sdASO delivery is a three-step process:

01Plate cells at optimal density (50-70% confluency at treatment)
02Add AUMlnc 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

Nuclear targeting: Unlike siRNAs, AUMlnc sdASO can effectively target nuclear-retained long non-coding RNAs (lncRNAs)
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 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

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 AUMlnc sdASO.
- Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay.
02
Step 2: 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 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: AUMlnc sdASO delivery to cells
Add AUMlnc 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 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 the three concentrations of the range (5 μM, 10 μM and 20 μM) to determine the optimal concentration for your specific lncRNA target.
- For some lncRNAs with complex secondary structures or nuclear localization, the top of the range, 20 μM, may be required for efficient knockdown.
Optimization tip: LncRNAs often have more complex secondary structures than mRNAs and may be less abundant. Starting at 10 μM and moving up may yield better results for many lncRNA targets compared to typical mRNA knockdown protocols.
Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.
04
Step 4: Incubation and analysis
Incubate cells with AUMlnc 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.
- Return cells to the incubator and maintain under standard culture conditions.
- Analyze AUMlnc sdASO-treated cells after the desired time point. Some nuclear lncRNAs may require 48-96 hours.
- 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.
Note: 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.
Return the plate to the humidified incubator under standard culture conditions, typically 24-72 hours, or 48-96 hours for a nuclear target.

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 AUMlnc 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 for lncRNA targeting

Target site selection

LncRNAs often have complex secondary structures. Target regions with predicted high accessibility.

Nuclear lncRNA considerations

For nuclear-retained lncRNAs, allow additional time (48-96 hours). 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 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 need more. Move up towards 20 μM for challenging lncRNA targets.

Extend incubation time: Nuclear lncRNAs may require 48-96 hours, due to nuclear accessibility and turnover rates.

Target accessibility: LncRNAs can have extensive secondary structures. Consider ordering alternative AUMlnc sdASOs 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 sdASOs are shipped in lyophilized form. Upon arrival, store at -20°C.
Resuspended AUMlnc 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 for lncRNA research

AUMlnc sdASOs reach the nucleus as well as the cytoplasm, where RNase H1-dependent cleavage acts on nuclear RNA in the same way. That is what puts a nuclear-retained lncRNA within reach, where a cytoplasmic RNAi pathway cannot follow it.
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 ordering 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.

Note

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

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A scientist reviews the target lncRNA, where it is retained and how the knockdown will be read before an order is placed.

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

AUMlnc™ sdASO™ protocol