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No. These products are self-delivering antisense oligonucleotides (sdASO™). Simply add them to your cell culture or inject in vivo – no lipids, electroporation, or viral delivery needed. This is a major advantage of AUM's technology. If you are using AUMsaver, then yes, you will need a transfection method (lipid-based is typical) because AUMsaver is not self-delivering.

Efficiency can vary by target, but typically we observe 50-95% mRNA knockdown for well-designed ASOs at optimal doses. Many of our case studies report >70% knockdown at the mRNA level and significant protein reductions. If you are consistently seeing <50%, please reach out; we might recommend an alternate target site or check for issues in your protocol. Sometimes extremely stable proteins won't show a full 50% decrease even if mRNA is down 80% (because the protein persists longer). In such cases, look at mRNA to gauge efficacy, and consider waiting longer or applying multiple doses.

AUMblock™ and AUMskip™ are specialized members of our sdASO™ portfolio designed for specific RNA modulation rather than degradation:

• Use AUMblock™ when you want to block specific RNA-protein interactions or regulatory sites without degrading the target RNA. AUMblock™ is designed with chemical modifications that prevent RNase H recruitment, allowing it to bind to its target and block access of proteins or other regulatory factors. It's ideal for studying RNA-binding protein interactions, blocking miRNA binding sites on mRNAs, or preventing regulatory complexes from accessing functional RNA domains. Unlike AUMsilence™ which degrades the target RNA, AUMblock™ preserves the RNA while selectively inhibiting specific interactions.
• Use AUMskip™ when you want to modulate pre-mRNA splicing, such as promoting exon skipping or inclusion. AUMskip™ is optimized for binding to splice sites or splicing regulatory elements, allowing you to manipulate which exons are included in the mature mRNA. This is particularly valuable for studying alternative splicing mechanisms, correcting disease-causing splice defects, or generating specific protein isoforms. AUMskip™ has been successfully used in models of neuromuscular disorders, cancer, and other diseases where splicing modulation is therapeutically relevant.

Both products utilize our self-delivering technology, so no transfection reagents are required. Their chemical modifications are specifically optimized for their respective functions – AUMblock™ for strong, stable binding without RNA degradation, and AUMskip™ for optimal interactions with splicing machinery and splice regulatory elements.

Consider your experimental model and budget. If working with primary cells, tough cell lines, or doing in vivo work – go with self-delivering ASOs (sdASO™) like AUMsilence, AUMantagomir, AUMlnc, AUMblock, or AUMskip for the convenience and higher likelihood of success in those systems. If you're working with an easy-to-transfect cell line (like HEK293, HeLa) and you need many different ASOs cost-effectively (e.g., a large screen), AUMsaver is a great choice to save on cost while still getting high potency knockdown. Some users even start with AUMsaver in cell line screens, then switch to sdASO™ products for validation in primary cells or animals. The sequences can be the same (just different levels of modification), so results are transferable.

Our ASOs are designed with AI-powered algorithms to minimize non-specific effects. Unlike siRNAs, they do not rely on the cell's microRNA machinery, so they won't saturate Dicer or RISC. Off-target binding to other mRNAs is unlikely unless there's a significant sequence complementarity which our AI design pipeline actively avoids. We do occasional whole-transcriptome expression checks in-house when developing ASOs and typically find that aside from the target gene, no other genes significantly change (except those downstream in pathway, which is the intended biological effect). However, one should always use a control to account for any slight interferon response or general effect if present – e.g., measure if ISG15, OAS genes are induced (they're usually not with our chemistry). If you see changes in those, it might indicate an immune response, in which case contact us; it could be sequence-dependent and we can tweak the design.

Yes, experiment design should be tailored to your specific RNA target:

• For mRNA targets (AUMsilence™): Standard knockdown experiments typically show effects within 24-72 hours. Measure both mRNA levels (by qRT-PCR) and protein levels (by Western blot or immunofluorescence) to assess knockdown efficiency. Include a time-course if protein half-life is unknown.
• For miRNA targets (AUMantagomir™): After treatment, measure both the target miRNA levels (by qRT-PCR) and the de-repression of known miRNA target genes. Since miRNAs often have subtle effects on multiple targets, consider transcriptome or proteome analysis to capture the full impact.
• For lncRNA targets (AUMlnc™): Since many lncRNAs function in the nucleus, consider nuclear fractionation to confirm knockdown in the relevant cellular compartment. Also assess impact on any known lncRNA-interacting proteins or regulated genes.
• For steric blocking (AUMblock™): Standard knockdown assays aren't appropriate since the RNA isn't degraded. Instead, measure functional outcomes like RNA-protein interactions (by RIP, CLIP), translation efficiency, or downstream pathway activation.
• For splice modulation (AUMskip™): Use PCR primers spanning the target exon(s) to measure splice isoform ratios. Also confirm altered protein variants by Western blot with antibodies recognizing domains affected by the splicing change.

For all targets, include appropriate controls (scrambled sequences, untreated cells) and consider dose-response experiments to determine optimal concentration.

There are a few possibilities:

• Delivery issue: If using AUMsaver, perhaps the transfection failed – include a control (like a fluorescent oligo or siGLO) to ensure delivery actually happened. If using self-delivering sdASO™ and in a very short time frame, maybe not enough uptake – usually by 24h we see effects though. Ensure you gave enough time and appropriate concentration.
• Incorrect sequence or dilution error: Double-check you added the correct ASO and amount. If you accidentally used scramble or too low a dose, that would explain it.
• Target not expressed: Make sure the cell type actually expresses the RNA you think. If baseline expression is zero or extremely low, "no change" doesn't mean ASO failed – it means there was nothing to knock down. Check baseline mRNA levels (qPCR Ct values) in your untreated vs treated to ensure the assay is working.
• ASO design issue: It's possible (though rare with our AI-optimized designs) that an ASO sequence is not effective due to target accessibility issues. If everything else is correct, we might provide an alternative sequence to try. We often design two sequences in tricky cases to have a backup.
• Sample handling: Ensure that when harvesting RNA, the process is effective. If you have RNase contamination or poor lysis, maybe the qPCR isn't reliable. Use proper RNA extraction kits and check RNA integrity if suspect.
• Product selection: If you're using AUMblock™ or AUMskip™, remember these are designed for modulation rather than degradation. AUMblock™ won't show mRNA knockdown in standard qPCR (it blocks interactions without degrading RNA), and AUMskip™ affects splicing patterns rather than total RNA levels. Use appropriate assays like splicing-sensitive PCR or protein binding assays to detect their effects.

If troubleshooting doesn't reveal the cause, please contact us – we'll walk through your experiment details to pinpoint the problem and provide a solution (like a replacement ASO or protocol tweak). Our goal is your success, and we will support until it works.

While AUM's sdASO™ products are designed to minimize toxicity, several factors could cause unexpected cell stress:

• Concentration too high: Try reducing the ASO concentration. While many cell types tolerate up to 10 μM, some sensitive cells may require lower doses (500 nM to 1 μM).
• Essential gene targeting: If your target gene is essential for cell survival or proliferation, knockdown itself might cause apparent "toxicity." This is actually a successful experiment showing the gene's importance. Include a non-targeting control ASO to distinguish between specific knockdown effects and non-specific toxicity.
• Cell density issues: Cells that are too sparse at treatment time may be more sensitive. Ensure cells are at 40-60% confluency when adding ASOs.
• Media conditions: Some sensitive cell types may respond better when ASOs are added in fresh, complete media rather than directly to existing media.
• Transfection reagent toxicity: If using AUMsaver with transfection reagents, the lipid carrier itself might be toxic. Optimize the transfection protocol by reducing reagent amount while maintaining delivery efficiency.
• Off-target effects: In rare cases, a specific ASO sequence might have unexpected off-target effects. Try an alternative ASO targeting a different region of the same gene.

If toxicity persists after these adjustments, please contact our scientific support team. We can help troubleshoot or provide alternative ASO designs optimized for sensitive cell models.

Inconsistent qPCR results can stem from several sources:

• RNA quality issues: Poor or variable RNA quality is a common source of inconsistency. Use a high-quality RNA isolation method and check RNA integrity (RIN score) if possible. Avoid freeze-thaw cycles of RNA samples.
• Inconsistent ASO delivery: For sdASO™ products, ensure thorough mixing when adding to culture medium. For AUMsaver with transfection, standardize your transfection protocol, including cell density, reagent amounts, and complex formation time.
• Variable cell conditions: Start with consistent cell density and passage number. Variations in cell confluence can affect gene expression and ASO uptake.
• qPCR technique: Use technical replicates for qPCR, carefully validated primers, and multiple reference genes for normalization. Ensure consistent reverse transcription efficiency between samples.
• Target gene variability: Some genes naturally fluctuate in expression due to cell cycle, stress responses, or other factors. In such cases, increased biological replicates and careful experimental timing can help.
• Primer design issues: Ensure your primers don't overlap with the ASO binding site, as this could interfere with accurate quantification. Design primers that amplify a region distinct from the ASO target site.

For the most consistent results, standardize all aspects of your workflow from cell plating to RNA isolation to qPCR setup. If inconsistency persists, consider using our fluorescently labeled ASOs to monitor uptake efficiency across different experiments.

This discrepancy between mRNA and protein knockdown is common and can be due to several factors:

• Protein half-life: The most common explanation is that your target protein has a long half-life. While mRNA levels drop quickly after ASO treatment, existing proteins persist until they naturally degrade. For stable proteins, this can take days or even weeks. Extend your time course to see if protein levels eventually decrease.
• Alternative translation mechanisms: Some genes have alternative translation initiation sites or can be translated from related transcripts that your ASO doesn't target. This allows continued protein production despite knockdown of the main mRNA.
• Feedback upregulation: Sometimes, cells compensate for reduced mRNA by increasing translation efficiency or stabilizing the remaining protein, partially counteracting the knockdown effect.
• Protein detection issues: Antibody specificity problems or western blot technical issues can mask actual protein reduction. Validate your antibody and consider alternative detection methods.
• Post-transcriptional regulation: MicroRNAs or RNA-binding proteins might be regulating your protein of interest, creating a disconnect between mRNA and protein levels.

To address this issue, try extending your time course, increasing ASO concentration (within non-toxic range), using multiple treatments over several days, or targeting multiple regions of the same gene simultaneously with a combination of ASOs. For particularly challenging targets, consider using AUMblock™ to directly interfere with protein translation rather than relying on mRNA degradation.

While our sdASO™ products are designed for efficient uptake without transfection reagents, you can optimize several factors to improve delivery:

• Optimize concentration: Try a range of concentrations (500 nM to 10 μM) to find the sweet spot for your specific cell type. Some cells require higher concentrations for optimal uptake.
• Cell density considerations: Uptake efficiency can vary with cell density. For adherent cells, aim for 30-60% confluency at treatment time. For suspension cells, a density of 0.5-1 × 10^6 cells/mL often works well.
• Serum factors: In some rare cases, certain serum components can affect uptake. Try treating cells in serum-free or reduced-serum media for 4-6 hours before returning to complete media (if your cells tolerate this).
• Multiple treatments: For difficult-to-transfect cells, consider a second treatment 24-48 hours after the first to boost overall uptake.
• Media pH and composition: Uptake mechanisms can be sensitive to pH and ion concentrations. Ensure your media is fresh and at the correct pH.
• Cell health and metabolism: Actively dividing cells often take up ASOs more efficiently than quiescent cells. Ensure your cells are in optimal growth conditions.

If you're still experiencing uptake challenges, consider using our fluorescently labeled ASOs to directly monitor cellular uptake and optimize conditions. For extremely resistant cell types (rare but possible), you can combine our sdASO™ products with commercial transfection reagents - they're fully compatible - though this is rarely necessary.

Splice modulation with AUMskip™ can be complex due to the intricate nature of splicing regulation. Here are key factors to troubleshoot:

• Target site accessibility: The splice site or splicing regulatory element might be less accessible than predicted. Try targeting alternative sites around the exon-intron junction, including exonic splicing enhancers (ESEs) or intronic splicing silencers (ISSs).
• Detection method: Standard qPCR may not detect splicing changes. Use RT-PCR with primers spanning multiple exons, followed by gel electrophoresis to visualize alternative splice products. Sequencing the PCR products can confirm exact splice junctions.
• Concentration and timing: Splice modulation often requires higher ASO concentrations than mRNA knockdown (5-20 μM). Also, the timing can differ - check multiple timepoints from 24-96 hours.
• Cell-specific splicing factors: Splicing regulation varies across cell types due to different splicing factor expression. Ensure your cell model expresses the necessary machinery for the specific splicing event you're studying.
• Competing splice enhancers/silencers: Multiple regulatory elements may control a single splicing event. You might need to target multiple sites simultaneously with a cocktail of AUMskip™ ASOs.
• Pre-mRNA processing kinetics: Splicing occurs co-transcriptionally and can be rapid. Ensure your AUMskip™ ASO has sufficient nuclear localization to interact with pre-mRNA before splicing occurs.

For challenging splice modulation, consider consulting with our scientific team for customized design strategies. We can develop optimized AUMskip™ ASOs based on your specific target and splicing context, incorporating factors like secondary structure prediction and splicing factor binding site analysis.

Control ASOs should ideally have minimal effects, but occasionally you might observe unexpected activity. Here's what to check:

• Sequence specificity: Even negative control ASOs can sometimes have partial complementarity to unintended targets. If using your own control sequence, check for potential off-targets using BLAST or similar tools. AUM's catalog control ASOs are extensively validated to minimize such effects.
• Non-specific cellular responses: At very high concentrations (>20 μM), any oligonucleotide can trigger mild stress responses in some cell types. Keep controls at the same concentration as your experimental ASOs, ideally ≤10 μM.
• Transfection-related effects: If using AUMsaver with transfection reagents, the reagent itself might affect gene expression or cell behavior. Include a mock transfection control (reagent only, no ASO).
• Technical variability: What appears as "knockdown" with a control ASO might be normal variability in your assay. Establish the baseline variability of your measurement system with multiple replicates of untreated samples.
• Activation of innate immune responses: Certain sequence motifs can trigger TLR activation or interferon responses. Our standard controls avoid immunostimulatory motifs, but custom sequences might inadvertently include them.

To address control ASO effects: (1) Validate multiple control sequences to find one with minimal impact on your system; (2) Include both untreated and control ASO-treated groups in your analysis; (3) Consider lowering your ASO concentration if effects persist at high doses; (4) Report any persistent issues to our scientific support team - we can provide alternative control sequences or troubleshooting assistance.