Protocol

AUMlnc toASO protocol

In vitro protocol for transfection-optimized antisense oligonucleotides targeting long non-coding RNAs

AUMlnc toASO are transfection-optimized antisense oligonucleotides for the knockdown of long non-coding RNAs in mammalian cell culture. They are the same designs at a lower price, delivered with a transfection reagent, and are used for high-throughput screening and routine lncRNA knockdown in standard cell lines.

Protocol overview

AUMlnc toASO delivery requires standard transfection methods. This protocol covers several approaches:

01Lipid-based transfection (most common method)
02Electroporation (for difficult-to-transfect cells)
03Calcium phosphate transfection (economical option)
04Polymer-based transfection

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

Key advantages

Cost-effective: A lower price than the self-delivering products, 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 specificity and the stability of AUMlnc sdASO

Materials and reagents

Required items

AUMlnc 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

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 transfection is typical.
- For adherent cells: Plate cells the day before transfection, at a seeding density that brings them to that confluency by the time of transfection. Allow cells to adhere overnight.
- For suspension cells: Prepare cells at the appropriate density shortly before transfection with AUMlnc toASO.
- Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay.
02
Step 2: AUMlnc toASO stock preparation
Prepare AUMlnc 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 AUMlnc 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.
- Prepare two working stocks from that one by diluting aliquots in sterile nuclease-free water, because a reconstituted stock is too concentrated to pipette accurately at the volumes a well needs at 50 nM to 100 nM. A 2 μM stock, a 1 in 50 dilution of it, for the lipid-based route, where the oligonucleotide is diluted into serum-free medium. And a 25 μM stock, a 1 in 4 dilution, for the calcium phosphate and polymer routes, where it goes into a small fixed volume whose proportions the chemistry depends on.
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.
03
Step 3: Transfection method selection
AUMlnc 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.
Selection tip: 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.
04
Step 4: Lipid-based transfection protocol
This protocol describes a general lipid-based transfection method for AUMlnc toASO. Always refer to the specific instructions provided by the manufacturer of your transfection reagent.
- Dilute AUMlnc toASO: In a sterile tube, dilute AUMlnc toASO in serum-free medium to achieve the desired concentration. For a 24-well plate, use 50 μL of serum-free medium and 15-30 μL of the 2 μM working stock, which gives a final concentration in the well of 50 nM to 100 nM, the recommended range.
- 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 AUMlnc 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.
Note: 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.
05
Step 5: Electroporation protocol
Electroporation is an effective method for delivering AUMlnc 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 AUMlnc toASO: Add AUMlnc toASO to the cell suspension to achieve a concentration of 1-2 μM in the electroporation buffer. 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.
Important: Electroporation parameters vary significantly between cell types and electroporation systems. Always optimize the electroporation conditions for your specific cells and equipment.
06
Step 6: 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 AUMlnc toASO (1.1-2.2 μL of the 25 μM working 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). It is the same volume as Solution A, which is what brings the 2X HBS to 1X where the precipitate forms; move one of the two and the other moves with it.
- 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.
Note: The pH of the 2X HBS is critical for efficient transfection. Small variations in pH can significantly affect transfection efficiency. So is the 1X the equal volumes give it, and so is the calcium: at the volumes above the oligonucleotide is <10% of Solution A and the mix carries about 57 to 60 mM calcium.
07
Step 7: Polymer-based transfection protocol
Polymer-based transfection is an alternative method that works well for certain cell types.
- Dilute AUMlnc toASO: In a sterile tube, dilute AUMlnc toASO in the buffer recommended by the polymer transfection reagent manufacturer. For a 24-well plate, use 50 μL of buffer and 1.1-2.2 μL of the 25 μM working stock, which keeps the oligonucleotide a small part of that buffer rather than a third of it.
- 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 AUMlnc 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.
Important note: Always follow the specific instructions provided by the manufacturer of your polymer-based transfection reagent, as protocols can vary significantly between products.
08
Step 8: Incubation and analysis
Incubate cells with AUMlnc toASO and analyze knockdown at appropriate time points.
- Return cells to the incubator and maintain under standard culture conditions.
- Analyze AUMlnc toASO-treated cells after the desired time point, typically 24-72 hours post-transfection. 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.

Reference calculations

Making the stock

Reconstitute the vial in nuclease-free water to 100 μM (10 μL per nmol, so 50 μL for a 5 nmol vial). Prepare the 2 μM and 25 μM working stocks from it (Step 2).

Vial	For 100 μM
2 nmol	20 μL
5 nmol	50 μL
10 nmol	100 μL
25 nmol	250 μL

Volumes for lipid-based transfection

Cell culture plate	96-well	24-well	12-well	6-well
AUMlnc toASO (2 μM working stock)¹	3-6 μL	15-30 μL	30-60 μL	63-125 μL
Serum-free medium for ASO dilution	10 μL	50 μL	100 μL	250 μL
Transfection reagent²	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	1 mL	2 mL

Table notes

These volumes give 50 nM to 100 nM in the well (the complete medium plus the two dilutions). The working stock and the reagent add a little volume, which lowers the final concentration by a few per cent.
The amount of transfection reagent can vary significantly depending on the specific product and cell type. Always refer to the manufacturer's recommendations.

Transfections per vial

At 100 nM, a 5 nmol vial transfects 416 wells of a 96-well plate, 83 of a 24-well plate or 41 of a 12-well plate. The volume in the well is the complete medium plus the two dilutions. Counts are rounded down to whole wells.

Plate	Volume in the well	50 nM	100 nM
96-well	120 μL	833	416
24-well	600 μL	166	83
12-well	1,200 μL	83	41
6-well	2,500 μL	40	20

Cell density at transfection

Transfect adherent cells with AUMlnc toASO when they 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

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, 50-70% 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.

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, cells treated with transfection reagent only, scrambled/non-targeting AUMlnc toASO, and positive control ASOs. For lncRNAs with adjacent or overlapping coding genes, carefully monitor effects on neighboring genes to confirm specificity.

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 50-70%) 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.

Move within the recommended range: The recommended final concentration in the well is 50-100 nM. If the first attempt was at 50 nM, raise it to the top of that range before changing anything else. If knockdown is still poor there, the thing to reconsider is the design rather than the dose: try a second sequence against the target.

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 be a consequence of successful knockdown.

Low knockdown efficiency

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 toASOs 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 toASO are shipped in lyophilized form. Upon arrival, store at -20°C.
Resuspended AUMlnc 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

AUMlnc toASO are optimized for standard transfection methods but do not self-deliver like AUMlnc sdASO.
Always follow the specific instructions provided by the manufacturer of your transfection reagent, as protocols can vary significantly between products.
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 toASO are for research use only. Not for use in diagnostic or therapeutic procedures.

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

AUMlnc™ toASO™ protocol