Controls and Validation Strategies for sdASO™ Products

Introduction

Proper experimental controls and validation methods are critical for generating reliable and publishable results with antisense oligonucleotides. This guide outlines recommended controls and validation strategies for all AUM Biotech sdASO™ products, including AUMsilence™, AUMantagomir™, AUMlnc™, AUMsilence V+™, AUMblock™, and AUMskip™, to ensure your gene silencing experiments produce robust, reproducible data.

Why Proper Controls Matter

Antisense oligonucleotide experiments require thoughtful controls to distinguish specific effects from non-specific or off-target effects. Well-designed controls help:

Confirm that observed phenotypic changes are due to target gene silencing
Rule out non-specific effects of oligonucleotide chemistry or delivery
Ensure experimental reproducibility and publication quality
Validate the specificity of your sdASO™
Provide confidence in your research conclusions

Best Practice

Always include both positive and negative controls in your sdASO™ experiments. For high-profile publications, consider including multiple validation methods to provide compelling evidence of specific gene silencing.

For larger studies targeting multiple genes, we recommend including controls for each experimental batch to account for any variation in experimental conditions.

Recommended Experimental Controls

Negative Controls

Negative controls are essential to establish baseline conditions and rule out non-specific effects of oligonucleotide treatment.

Untreated Control: Cells or samples that receive no treatment. This control establishes the baseline expression of your target gene and cellular phenotype.

Non-targeting Control sdASO™: AUM Biotech offers non-targeting (scrambled) sdASO™ controls designed with similar chemical composition but without complementarity to any known mammalian transcript. These controls help distinguish between specific knockdown effects and non-specific effects of oligonucleotide chemistry.

Mismatch Control sdASO™: For the most rigorous experiments, consider using a mismatched control—an sdASO™ with 2-5 nucleotide mismatches to your target sequence. This helps assess sequence specificity of target recognition.

Note

Non-targeting control sdASO™ should be used at the same concentration as your experimental sdASO™. For dose-response studies, include controls at each concentration tested.

Positive Controls

Positive controls confirm that your experimental system is functioning properly and provide a reference for expected knockdown efficiency.

Housekeeping Gene Targeting sdASO™: AUM Biotech offers sdASO™ that target well-characterized housekeeping genes (e.g., GAPDH, β-actin, or HPRT) with predictable knockdown efficiency. These controls verify that your experimental conditions support effective gene silencing.

Well-validated Gene Target: If available for your model system, include an sdASO™ targeting a gene with well-characterized knockdown phenotype in your cell type or model organism.

Important

When using housekeeping genes as positive controls, be aware that their knockdown may affect cell viability or physiology. For long-term experiments, consider using a positive control targeting a non-essential gene.

Phenotype Rescue Controls

Rescue experiments provide the strongest evidence that observed phenotypes are specifically due to knockdown of your target gene rather than off-target effects.

Overexpression Rescue: Re-introduce an RNAi-resistant version of your target gene (containing silent mutations at the sdASO™ binding site) to see if it rescues the knockdown phenotype.

Multiple sdASO™ Targeting: Use two or more sdASO™ targeting different regions of the same transcript. If they produce the same phenotype, it strongly suggests the effect is due to specific target knockdown.

Small Molecule Rescue: For some pathways, small molecule activators or inhibitors can be used to rescue or mimic the knockdown phenotype, providing orthogonal validation.

Time Point and Dose Controls

These controls help establish the temporal dynamics of knockdown and dose-response relationship.

Time Course: Collect samples at multiple time points (e.g., 24h, 48h, 72h, 96h) after sdASO™ treatment to determine the optimal time for analysis of knockdown and downstream effects.

Dose Response: Test multiple sdASO™ concentrations (e.g., 500 nM, 1 μM, 5 μM, 10 μM) to establish the dose-response relationship and identify the optimal concentration for your specific target and cell type.

Optimization Tip

The timing of knockdown assessment is critical. mRNA levels typically decrease before protein levels. Consider the half-life of your target protein when designing time point controls.

Control Selection Guide

Use this table to select the appropriate controls based on your experimental goals:

Experimental Goal	Essential Controls	Recommended Additional Controls
Basic target validation	Untreated control; Non-targeting sdASO™	Time course; Dose response
Publication-quality research	Untreated control; Non-targeting sdASO™; Positive control sdASO™	Multiple sdASO™ targeting different regions; Mismatch control sdASO™
Mechanistic studies	Untreated control; Non-targeting sdASO™; Positive control sdASO™	Rescue experiment; Multiple validation methods
Therapeutic target validation	Untreated control; Non-targeting sdASO™; Multiple sdASO™ targeting same gene	Rescue experiment; Small molecule validation

Validation Methods for sdASO™ Knockdown

Validation is the process of confirming that your sdASO™ effectively reduces the expression of your target gene. Multiple validation methods are recommended for comprehensive analysis.

mRNA Level Validation

Quantifying target mRNA levels is the most direct method to assess knockdown efficiency of sdASO™ products.

RT-qPCR (Recommended): Real-time quantitative PCR is the gold standard for mRNA quantification.

Extract total RNA from treated and control samples
Synthesize cDNA using reverse transcriptase
Perform qPCR with primers flanking or spanning the sdASO™ binding site
Normalize to stable reference genes (at least 2-3 reference genes recommended)
Calculate relative expression using the ΔΔCt or standard curve method

Northern Blot: Though less common, northern blotting can provide visual confirmation of target mRNA reduction and detect any degradation products.

RNA-Seq: For transcriptome-wide analysis, RNA-Seq can validate target knockdown while also revealing effects on related pathways and potential off-targets.

Note

When designing RT-qPCR primers, ensure they are outside the sdASO™ binding region to avoid interference from bound sdASO™. For exon-skipping applications (AUMskip™), design primers to specifically detect the altered splice variant.

Protein Level Validation

Protein-level validation confirms that mRNA knockdown translates to reduced protein expression, which is particularly important for functional studies.

Western Blot: The most common method for protein-level validation.

Extract protein from treated and control samples
Separate by SDS-PAGE and transfer to membrane
Probe with specific antibodies against your target protein
Use appropriate loading controls (e.g., GAPDH, β-actin, tubulin)
Quantify band intensity using image analysis software

Immunofluorescence/Immunocytochemistry: Provides spatial information about protein expression.

Fix cells and perform antibody staining for your target protein
Include appropriate controls (primary antibody omission, isotype controls)
Analyze by fluorescence microscopy or high-content imaging

Flow Cytometry: For targets expressed on the cell surface or when using intracellular staining.

ELISA: For secreted proteins or when quantitative measurement is needed.

Timing Tip

Protein knockdown typically lags behind mRNA knockdown. Consider the half-life of your target protein when scheduling protein-level validation. For proteins with long half-lives (several days), extend your time course to capture maximal protein reduction.

Functional Validation

Functional assays confirm that target knockdown leads to the expected biological consequences and provide insight into gene function.

Phenotypic Assays: Select assays relevant to your target's function:

Proliferation/viability assays (e.g., MTT, XTT, CellTiter-Glo)
Migration/invasion assays
Differentiation assays
Reporter gene assays
Pathway-specific assays (e.g., signaling pathway activation)

Rescue Experiments: Re-introducing the target gene or activating the pathway downstream of your target should reverse the knockdown phenotype if effects are specific.

Downstream Target Analysis: Measure known downstream effectors of your target to confirm pathway modulation:

For transcription factors: measure expression of known target genes
For signaling proteins: assess phosphorylation status of pathway components
For enzymes: measure substrate or product levels

Product-Specific Validation Strategies

Different AUM Biotech products require specialized validation approaches based on their targets and mechanisms.

For AUMsilence™ (mRNA targeting): Standard RT-qPCR and Western blot validation is typically sufficient.

For AUMantagomir™ (miRNA inhibition):

miRNA quantification (qPCR, Northern blot, or small RNA-Seq)
De-repression of known miRNA target genes (increased expression)
miRNA target reporter assays (luciferase reporters containing miRNA binding sites)

For AUMlnc™ (lncRNA targeting):

lncRNA quantification by RT-qPCR
RNA FISH for nuclear lncRNAs
Assessment of known lncRNA-dependent processes

For AUMskip™ (exon skipping):

RT-PCR with primers flanking the skipped exon to visualize splice variants
Western blot to confirm production of the altered protein isoform
Functional assays specific to the protein isoform

For AUMblock™ (steric blocking):

Protein expression analysis (as the RNA remains intact but function is blocked)
Splicing analysis for splice-modulating applications
RNA-protein interaction assays if targeting RNA-protein binding sites

For AUMsilence V+™ (viral RNA targeting):

Viral RNA quantification (RT-qPCR)
Viral protein expression (Western blot, immunofluorescence)
Viral titer or plaque assays
Viral replication assays

Validation Method Selection Guide

Use this table to select the appropriate validation methods based on your sdASO™ product and application:

sdASO™ Product	Primary Validation	Secondary Validation	Functional Validation
AUMsilence™	RT-qPCR for target mRNA	Western blot for target protein	Phenotypic assays relevant to target function
AUMantagomir™	miRNA qPCR	De-repression of miRNA targets (mRNA/protein upregulation)	miRNA reporter assays; pathway analysis
AUMlnc™	RT-qPCR for lncRNA	RNA FISH or subcellular fractionation + RT-qPCR	Assessment of lncRNA-dependent processes
AUMblock™	Target function assessment	RNA binding/structural assays	Pathway-specific functional assays
AUMskip™	RT-PCR for splice variants	Western blot for protein isoforms	Isoform-specific functional assays
AUMsilence V+™	Viral RNA RT-qPCR	Viral protein expression	Viral titer or infectivity assays

Data Analysis and Interpretation

Quantifying Knockdown Efficiency

Properly quantifying and reporting knockdown efficiency is essential for comparing results across experiments and publications.

mRNA Knockdown: Calculate percent knockdown relative to control samples:
% Knockdown = (1 - Relative Expression) × 100%
Protein Knockdown: Normalize to loading controls and calculate percent reduction compared to control samples
Statistical Analysis: Perform appropriate statistical tests to determine significance:
- t-test for comparing two groups
- ANOVA for comparing multiple groups
- Include p-values and error bars in figures
Biological Replicates: Perform at least 3 independent biological replicates for reliable statistical analysis

Interpreting Results

Consider these factors when interpreting your knockdown and validation data:

Expected Knockdown Range: Typically 60-95% mRNA reduction with sdASO™ products, though this varies by target, cell type, and concentration
mRNA vs. Protein Discrepancy: Protein reduction may be less pronounced than mRNA reduction due to protein stability or compensatory mechanisms
Biological Significance: Even partial knockdown may be sufficient to observe phenotypic effects for some targets
Cell-to-Cell Variability: Consider using single-cell methods if population heterogeneity is a concern
Temporal Dynamics: Track knockdown over time to identify optimal windows for downstream assays

Data Reporting Tip

When reporting knockdown efficiency in publications, always specify:

The method used for quantification (e.g., RT-qPCR, Western blot)
The time point at which knockdown was measured
The sdASO™ concentration used
The reference genes or loading controls used for normalization
The number of biological replicates

Tips and Troubleshooting

Best Practices for Successful Validation

Multiple Validation Methods

Validate knockdown at both mRNA and protein levels when possible. For high-impact research, include functional validation to demonstrate biological consequences.

Primer Design for RT-qPCR

Design primers that amplify a region outside the sdASO™ binding site to avoid potential interference. When possible, use primers that span exon-exon junctions to avoid genomic DNA amplification.

Reference Gene Selection

Validate the stability of reference genes under your experimental conditions. The ideal reference genes should be unaffected by your treatment. Using multiple reference genes (2-3) improves reliability.

Antibody Validation

Verify antibody specificity for protein-level validation. Consider using multiple antibodies targeting different epitopes or validating specificity with a knockout/knockdown control.

Troubleshooting Common Validation Issues

Insufficient mRNA Knockdown

Increase sdASO™ concentration: Try higher concentrations (5-20 μM) for difficult-to-silence targets.
Extend treatment time: Evaluate knockdown at later time points (72-96 hours).
Check RNA quality: Poor RNA quality can affect RT-qPCR results. Verify RNA integrity using an Agilent Bioanalyzer or gel electrophoresis.
Redesign sdASO™: The target region may be inaccessible due to RNA structure or protein binding. Contact AUM Biotech for design of alternative sdASO™ targeting different regions.
Verify target expression: Confirm that your target is expressed in your cell model under your experimental conditions.

Good mRNA Knockdown but Poor Protein Reduction

Extend the time course: Proteins with long half-lives require extended time for observable reduction. Consider assessing protein levels at later time points (72-120 hours).
Check protein half-life: Literature search for your protein's half-life can help set appropriate expectations.
Verify antibody quality: Poor antibody specificity or sensitivity can affect Western blot results.
Consider proteasome inhibition: If your protein is rapidly degraded, briefly treating with a proteasome inhibitor before lysis might help detect low-abundance proteins.
Assess post-transcriptional regulation: Some proteins are regulated at the translational level or have compensatory mechanisms that maintain protein levels despite reduced mRNA.

Inconsistent Knockdown Between Experiments

Standardize experimental conditions: Maintain consistent cell density, passage number, and treatment protocols.
Prepare consistent sdASO™ stocks: Aliquot stocks to avoid multiple freeze-thaw cycles.
Control for cell confluency: Extremely high or low cell confluency can affect sdASO™ uptake and activity.
Check for mycoplasma contamination: Contamination can alter cell physiology and affect experimental outcomes.
Standardize validation protocols: Use consistent RNA/protein extraction methods, RT-qPCR protocols, and analysis procedures.

Unexpected Phenotypic Results

Verify knockdown specifically correlates with phenotype: Use multiple sdASO™ targeting different regions of the same gene to confirm specificity.
Consider compensatory mechanisms: Cells may activate alternative pathways to compensate for the loss of your target gene.
Evaluate potential off-target effects: Use appropriate controls (non-targeting, mismatch) and rescue experiments.
Assess cell health: Ensure that observed phenotypes are not due to general cell stress or toxicity.
Consider cell type differences: Gene function may vary between cell types due to different pathway components or cellular context.

Additional Considerations

Considerations for In Vivo Validation

Tissue Distribution Analysis: Assess sdASO™ biodistribution using fluorescently labeled ASOs or tissue RT-qPCR
Tissue-specific Knockdown: Validate target reduction in the tissues of interest, not just in whole animal
Dosing Optimization: Perform dose-response studies to determine optimal dosing regimen
Serum Stability: Consider measuring sdASO™ levels in serum to assess stability in vivo
Multi-level Validation: Include molecular (RNA/protein), cellular, and physiological endpoints
Off-target Screening: Include additional controls for potential immune responses to the sdASO™

Long-term and High-throughput Applications

Sustained Knockdown: For long-term experiments, monitor knockdown persistence and re-dose as needed
Stable Cell Lines: Consider generating stable cell lines expressing inducible shRNAs for sustained knockdown
High-throughput Screening: For screening applications, use consistent positive and negative controls across all plates/batches
Multi-target Studies: When targeting multiple genes, include gene-specific validation for each target
Data Management: Implement robust data tracking and analysis systems for large-scale studies

Publication-Ready Validation

For journal submissions, especially in high-impact publications, reviewers often expect comprehensive validation. Consider including:

Both mRNA and protein level validation
Multiple independent sdASO™ sequences targeting the same gene
Rescue experiments
Dose-response and time-course data
Multiple negative controls (untreated, non-targeting, mismatch)

Need Help with Experimental Design or Validation?

Our scientific team is available to help you design optimal control and validation strategies for your specific experiment. We can recommend appropriate controls, validation methods, and troubleshooting approaches tailored to your research goals.

Request Control sdASO™Contact Scientific Support

Related Resources

AUMsilence™ Protocol

Detailed protocol for mRNA knockdown using our self-delivering AUMsilence™ sdASO™.

View Protocol

RT-qPCR Primer Design Guide

Guidelines for designing optimal RT-qPCR primers for sdASO™ validation.

View Guide

FAQs

Find answers to frequently asked questions about controls and validation for sdASO™ experiments.

View FAQs