Controls and validation strategies

1. 01

   Step 1: 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.

2. 02

   Step 2: 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.

3. 03

   Step 3: 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.
4. 04

   Step 4: 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 the three concentrations of the range (5 μM, 10 μM and 20 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:

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

1. 01

   Step 1: 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 most commonly used method 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 (AUMsplice™ sdASO™), design primers to specifically detect the altered splice variant.

2. 02

   Step 2: 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.

3. 03

   Step 3: 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
4. 04

   Step 4: Product-specific validation strategies

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

   • For AUMsilence sdASO (mRNA targeting): Standard RT-qPCR and Western blot validation is typically sufficient.
   • For AUMantagomir sdASO (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 sdASO (lncRNA targeting):
     • lncRNA quantification by RT-qPCR
     • RNA FISH for nuclear lncRNAs
     • Assessment of known lncRNA-dependent processes
   • For AUMsplice sdASO (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 sdASO (steric blocking):
     • Protein expression analysis (function is blocked rather than the RNA cleaved)
     • Splicing analysis for splice-modulating applications
     • RNA-protein interaction assays if targeting RNA-protein binding sites
   • For AUMsilence V+ sdASO (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:

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: 70-95% knockdown, measured as mRNA reduction against an untreated or non-targeting control. Knockdown is target and cell-type dependent.
• 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

Best practices for successful validation

Troubleshooting common validation issues

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 only 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

• AUMsilence™ sdASO™ protocol Detailed protocol for mRNA knockdown using our self-delivering AUMsilence™ sdASO™.
• FAQs Find answers to frequently asked questions about controls and validation for sdASO™ experiments.