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FAQ

FAQs and troubleshooting

Frequently asked questions

Do I need transfection reagents for AUMsilence sdASO, AUMantagomir sdASO, AUMlnc sdASO, AUMblock sdASO, or AUMsplice sdASO?
No. These products are self-delivering antisense oligonucleotides (sdASO). Add them to your cell culture or inject in vivo – no transfection reagent, electroporation, or viral vector needed. If you are using AUMsilence toASO, then yes, you will need a transfection method (lipid-based is typical) because AUMsilence toASO is not self-delivering.
What knockdown efficiency can I expect?
Efficiency can vary by target and cell type, but typically we observe 70-95% knockdown for well-designed ASOs at optimal doses. Many of our case studies report mRNA knockdown at that level and significant protein reductions. If you are consistently seeing less than that, please contact us; we might recommend an alternate target site or check for issues in your protocol. Sometimes extremely stable proteins will not 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.
When should I use AUMblock sdASO or AUMsplice sdASO instead of AUMsilence sdASO?
AUMblock and AUMsplice are members of our sdASO portfolio designed for specific RNA modulation rather than RNase H mediated degradation:
  • Use AUMblock sdASO when you want to block specific RNA-protein interactions or regulatory sites without recruiting RNase H to the target RNA. AUMblock sdASO is designed with chemical modifications that prevent RNase H recruitment, allowing it to hybridize to its target and block access of proteins or other regulatory factors. It is ideal for studying RNA-binding protein interactions, blocking miRNA binding sites on mRNAs, or preventing regulatory complexes from accessing functional RNA domains. Unlike AUMsilence sdASO which recruits RNase H1 to degrade the target RNA, AUMblock sdASO does not cleave it, and selectively inhibits specific interactions instead.
  • Use AUMsplice sdASO when you want to modulate pre-mRNA splicing, such as promoting exon skipping or inclusion. AUMsplice sdASO 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 in research models, or generating specific protein isoforms.
Both products use our self-delivering technology, so no transfection reagents are required. Their chemical modifications are specifically optimized for their respective functions – AUMblock sdASO for strong, stable hybridization without RNase H recruitment, and AUMsplice sdASO for interactions with splicing machinery and splice regulatory elements.
How do I choose between sdASO products and AUMsilence toASO?
Consider your experimental model and budget. If working with primary cells, difficult-to-transfect cell lines, or doing in vivo work – choose self-delivering ASOs (sdASO) like AUMsilence, AUMantagomir, AUMlnc, AUMblock, or AUMsplice for the convenience and higher likelihood of success in those systems. If you are working with an easy-to-transfect cell line (like HEK293, HeLa) and you need many different ASOs cost-effectively (e.g., a large screen), AUMsilence toASO is a great choice to save on cost while still getting high potency knockdown. Some users even start with AUMsilence toASO in cell line screens, then switch to sdASO products for validation in primary cells or animals. The sequences can be the same (different levels of modification), so results are transferable.
Will the ASO affect microRNA pathways or other genes non-specifically?
Our ASOs are designed to minimize non-specific effects. Unlike siRNAs, they do not rely on the cell's microRNA machinery, so they will not saturate Dicer or RISC. Off-target effects on other mRNAs are unlikely unless there is a significant sequence complementarity, and each sequence is checked for specificity against the transcriptome. 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 are typically 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 adjust the design.
Are there differences in how I should design experiments for various RNA targets?
Yes, experiment design should be tailored to your specific RNA target:
  • For mRNA targets (AUMsilence sdASO): 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 sdASO): 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 sdASO): 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 sdASO): Standard knockdown assays are not the primary readout, since AUMblock sdASO does not cleave the RNA. Instead, measure functional outcomes like RNA-protein interactions (by RIP, CLIP), translation efficiency, or downstream pathway activation.
  • For splice modulation (AUMsplice sdASO): 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.

Troubleshooting guide

Solutions to common technical challenges with RNA silencing experiments

Each entry opens on the symptom, then the causes worth checking. If none of them fits, the link at the foot of the page books a web meeting with a scientist.

My ASO treatment didn't cause any knockdown. What could be wrong?
There are a few possibilities:
  • Delivery issue: If using AUMsilence toASO, perhaps the transfection failed – include a control (like a fluorescent oligo) to ensure delivery happened. If using self-delivering sdASO and in a very short time frame, maybe not enough uptake. 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 expresses the RNA you think. If baseline expression is zero or extremely low, "no change" does not 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 is possible 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 2 sequences in difficult 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 is not reliable. Use proper RNA extraction kits and check RNA integrity if suspect.
  • Product selection: If you are using AUMblock sdASO or AUMsplice sdASO, remember these are designed for modulation rather than RNase H mediated degradation, so transcript abundance is not the primary readout for either. Use appropriate assays like splicing-sensitive PCR or protein binding assays to detect their effects. A fall in mRNA level does not mean the wrong product was used, since a blocked or redirected transcript may be degraded by the cell.
If troubleshooting does not reveal the cause, please contact us – we will work through your experiment details to pinpoint the problem and provide a solution (like a replacement ASO or protocol adjustment). Troubleshooting and scientific support is a web meeting with a scientist, who works through the product, the lot, the protocol step and what you observed.
I'm seeing high cellular toxicity after ASO treatment. What could be causing this?
While AUMsilence sdASOs are designed to minimize toxicity, several factors could cause unexpected cell stress:
  • Concentration too high: Try reducing the ASO concentration: move down towards 5 μM, the bottom of the recommended range, and lower still for a line that is still stressed there.
  • Essential gene targeting: If your target gene is essential for cell survival or proliferation, knockdown itself might cause apparent "toxicity." This is 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 50-70% 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 AUMsilence toASO 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.
My qPCR results show inconsistent knockdown between replicates. How can I improve reproducibility?
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 AUMsilence toASO 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 do not 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.
I see good mRNA knockdown but minimal protein reduction. Why might this happen?
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 does not 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 sdASO to directly interfere with protein translation rather than relying on RNase H mediated degradation.
How can I improve uptake of self-delivering ASOs in my cells?
While our sdASO products are designed to be taken up by endocytosis with no transfection reagent, you can optimize several factors to improve delivery:
  • Optimize 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.
  • Cell density considerations: Uptake efficiency can vary with cell density. For adherent cells, aim for 50-70% 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 more ASOs than quiescent cells. Ensure your cells are in optimal growth conditions.
If you are 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 are fully compatible), though this is rarely necessary.
AUMsplice sdASO isn't producing the expected splicing change. What should I check?
Splice modulation with AUMsplice sdASO 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 needs the upper end of the recommended range (5-20 μM) where a knockdown works from the starting point. Also, the timing can differ - check multiple timepoints from 24-96 hours after treatment.
  • 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 are 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 AUMsplice sdASOs.
  • Pre-mRNA processing kinetics: Splicing occurs co-transcriptionally and can be rapid. Ensure your AUMsplice sdASO 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 AUMsplice sdASOs based on your specific target and splicing context, incorporating factors like secondary structure prediction and splicing factor binding site analysis.
My control ASO is causing some knockdown or phenotypic effects. Is this normal?
Control ASOs should ideally have minimal effects, but unexpected activity is occasionally observed. Here is 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 AUMsilence toASO 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.
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.

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