T Cells RNA Silencing Guide

Master RNA Silencing in T Cells

Overcome transfection challenges with self-delivering ASOs

70-95%

Knockdown Efficiency

>95%

Cell Viability

None

Transfection Required

Why T Cells Are Challenging for Gene Silencing

T lymphocytes are critical orchestrators of adaptive immunity, but their unique biology makes them among the most challenging cell types for genetic manipulation. Unlike adherent cell lines, T cells exist in suspension with limited endocytic activity, particularly in their resting state.

The field has long struggled with fundamental barriers: lipofection reagents trigger activation-induced cell death (AICD) in activated T cells, electroporation causes significant mortality and phenotypic changes, and viral vectors raise safety concerns for clinical translation. Studies have shown that conventional transfection can reduce T cell viability to 40-60% within 24-48 hours while fundamentally altering cytokine production profiles.

AUM BioTech's self-delivering ASO technology solves these fundamental challenges. By leveraging gymnotic uptake—the spontaneous, energy-independent cellular entry of phosphorothioate-modified oligonucleotides—AUMsilence eliminates the need for transfection reagents entirely. This preserves T cell viability above 95% while maintaining normal activation states, proliferation capacity, and effector functions.

The scientific basis for self-delivering ASOs is well-established in the oligonucleotide therapeutic literature (Crooke et al., Nucleic Acids Research; Stein et al., Antisense Research). Phosphorothioate (PS) backbone modifications confer both nuclease resistance and the ability to cross cell membranes through interactions with cell surface proteins, enabling efficient delivery without artificial carriers.

T cells have low endocytic activity and resist conventional transfection

Lipofection causes activation-induced cell death (AICD) in activated T cells

Electroporation reduces viability and alters T cell phenotype and function

Primary T cells show high donor-to-donor variability with traditional methods

AUMsilence sdASO enables transfection-free knockdown via gymnotic delivery

Phosphorothioate modifications enable spontaneous cellular uptake

Why Conventional T Cell Transfection Methods Fail

T cells present unique biological barriers that cause conventional transfection methods to underperform or fail entirely:

Activation-Induced Cell Death (AICD)

Lipofection reagents trigger the Fas/FasL apoptosis pathway in activated T cells. Cationic lipids cause membrane stress that mimics activation signals, resulting in 40-60% cell death within 24-48 hours of transfection. This is particularly problematic for functional assays requiring viable effector T cells.

High Impact

Limited Endocytic Activity

Resting T cells have minimal endocytic uptake machinery. Activation with anti-CD3/CD28 increases endocytosis, creating a narrow 24-48h window for lipofection. Outside this window, transfection efficiency drops below 10-15%, making it impossible to study resting or memory T cell populations.

High Impact

Phenotype and Functional Alteration

Electroporation causes plasma membrane disruption that fundamentally alters T cell biology. Studies show altered calcium flux, changed cytokine secretion patterns (IL-2, IFN-γ, TNF-α), and reduced proliferative capacity. This makes functional readouts unreliable.

High Impact

Donor-to-Donor Variability

Primary human T cells from different donors exhibit 30-80% variation in transfection efficiency with conventional methods. This variability stems from differences in activation state, memory phenotype distribution, and intrinsic membrane properties, requiring extensive optimization for each donor.

Medium Impact

Clinical Translation Barriers

Viral vectors for CAR-T and adoptive cell therapies face regulatory scrutiny, high production costs, and potential insertional mutagenesis risks. Manufacturing timelines of 2-4 weeks for lentiviral production limit clinical scalability.

Medium Impact

Immunogenic Activation and Experimental Artifacts

Transfection reagents, particularly cationic lipids and viral vectors, trigger innate immune responses through TLR activation in T cells. This creates experimental artifacts where observed effects cannot be distinguished from transfection-induced activation. Pattern recognition receptor engagement alters baseline cytokine profiles, activation markers, and metabolic states, making it impossible to isolate target-specific phenotypes from delivery-induced perturbations.

High Impact

Method Comparison

Method	Efficiency	Viability	Pros	Cons
Lipofection (Cationic Lipid Reagents)	15-40%	40-60%	Simple protocol, commercially available	High toxicity, AICD, requires activation window, donor variability
Electroporation (Commercial Systems)	40-70%	50-75%	Higher efficiency than lipofection	Significant cell death, alters phenotype, requires specialized equipment, expensive consumables
Viral Vectors (Lentivirus, AAV)	70-90%	80-90%	High efficiency, long-term expression	Safety concerns, 2-4 week production, expensive, regulatory challenges, insertional mutagenesis risk
AUMsilence sdASO	70-95%	>95%	No transfection, preserves function, works in resting and activated cells, no equipment, reduced donor variability compared to transfection methods	Transient knockdown (appropriate for functional studies)

AUMsilence sdASO

The Gold Standard for T Cell Gene Silencing

Why This Product?

AUMsilence self-delivering ASOs are specifically designed to overcome the unique transfection challenges of T cells. Our phosphorothioate (PS) backbone modifications enable spontaneous cellular uptake via gymnotic delivery—eliminating lipofection-induced AICD, electroporation-mediated phenotype changes, and viral vector safety concerns.

Key Benefits

Zero Transfection Required

Simply add to culture medium. No lipids, no electroporation, no viral vectors. Eliminates AICD and preserves >95% viability.

70-95% Knockdown Efficiency

Robust, reproducible gene silencing across diverse T cell types: primary CD3+, CD4+, CD8+, Tregs, activated T cells, and cell lines (Jurkat, MOLT-4).

Preserved T Cell Function

Normal proliferation (CFSE dilution), cytokine production (IFN-γ, IL-2, TNF-α), and cytotoxic activity maintained. Ideal for functional assays.

Consistent Across Donors

Minimal donor-to-donor variability. Works without optimization across different PBMC donors, HLA types, and memory/naive subsets.

Rapid Timeline

No cloning, no virus production. From design to validated knockdown in 5-7 days. 48-72h post-treatment for mRNA knockdown, 72-96h for protein.

Scalable for CAR-T Manufacturing

Simple addition to culture—compatible with clinical-scale T cell expansion protocols. No specialized equipment required.

Ideal For

Primary human T cells (CD3+, CD4+, CD8+, Tregs)
CAR-T cell engineering and optimization
Tumor-infiltrating lymphocyte (TIL) therapy
Immune checkpoint silencing (PD-1, CTLA-4, LAG-3, TIM-3)
T cell exhaustion and dysfunction studies
Cytokine modulation (IL-2, IFN-γ, TNF-α, IL-10)
Transcription factor studies (FOXP3, T-bet, GATA3, RORγt)
Functional screening in Jurkat, MOLT-4, or other T cell lines
Co-culture and cytotoxicity assays

Learn More About AUMsilence sdASO

Alternative Products

AUMantagomir sdASO

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AUMlnc sdASO

When to use: For nuclear-retained long non-coding RNAs (lncRNAs) in T cell biology. Designed for targets not exported to cytoplasm.

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AUMsilence Protocols for T Cells

Cell-type-optimized protocols for primary T cells, T cell lines, and activated T cells. No transfection reagents required.

Quick Start Protocol (All T Cell Types)

Culture T cells at 0.5-1 × 10⁶ cells/mL in appropriate medium
Add AUMsilence sdASO directly to culture medium at 10 μM (5 μM may be used depending on cell type and target; higher concentrations may be needed for highly stable genes; no transfection reagent)
Incubate 48-72 hours at 37°C, 5% CO₂
Validate knockdown by qRT-PCR (mRNA) and flow cytometry or Western blot (protein)

Cell-Type-Specific Protocols

Primary Human T Cells (CD3+)

Optimal for freshly isolated PBMCs or cryopreserved T cells

T Cell Isolation

Isolate CD3+ T cells from PBMCs using negative selection (magnetic bead-based isolation kit). Negative selection preserves surface markers and activation state.

Materials: RPMI-1640 + 10% FBS + 1% Pen/Strep + 2 mM L-glutamine + 50 μM β-mercaptoethanol

Note: Avoid positive selection (anti-CD3 beads) if studying TCR signaling

Day 0

Cell Seeding

Seed T cells at 0.5-1 × 10⁶ cells/mL in 24-well plate (500 μL/well) or appropriate culture vessel. Use complete RPMI-1640 medium.

Materials: 24-well plate, complete RPMI-1640

Note: T cells prefer suspension culture at this density range

Day 0

AUMsilence Treatment

Add AUMsilence sdASO directly to culture medium at 10 μM final concentration (5 μM may be sufficient for some targets). Calculate volume: For 500 μL culture, add 5 μL of 1 mM AUMsilence stock. Mix gently by pipetting. Note: Optimal concentration typically ranges from 1-20 μM depending on cell doubling time and target gene stability.

Materials: AUMsilence sdASO (1 mM stock in nuclease-free water)

Note: No media change or washing required. Gymnotic uptake begins within 2-4 hours.

Day 0 or Day 1

Incubation

Incubate at 37°C, 5% CO₂ for 48-72 hours. Monitor cell density; split if exceeding 2 × 10⁶ cells/mL.

Materials: Humidified incubator

Note: Peak knockdown typically achieved at 48-72h. Can harvest earlier (24h) for time-course experiments.

Days 0-3

Validation

Harvest cells by centrifugation. For mRNA: qRT-PCR (expect 60-85% knockdown). For protein: flow cytometry (surface markers) or Western blot (intracellular proteins).

Materials: RNA extraction kit, qPCR reagents, antibodies

Note: Include viability staining (7-AAD or Live/Dead). Expect >95% viability.

Days 2-3

Activated T Cells (Anti-CD3/CD28)

For studying effector T cell function and cytokine production

T Cell Activation

Stimulate isolated T cells with plate-bound anti-CD3 (5 μg/mL) + soluble anti-CD28 (2 μg/mL) or magnetic CD3/CD28 activation beads (1:1 bead-to-cell ratio). Culture at 1 × 10⁶ cells/mL.

Materials: Anti-CD3/CD28 antibodies or activation beads, complete RPMI + IL-2 (50-100 U/mL)

Note: Activation increases metabolic activity and enhances ASO uptake

Day -1 (24h before ASO treatment)

AUMsilence Treatment (Post-Activation)

At 24h post-activation, add AUMsilence sdASO directly to activated T cells at 10 μM (5 μM may be sufficient depending on target; higher concentrations may be needed for highly stable genes). No media change needed. Beads remain in culture. Optimal concentration typically ranges from 1-20 μM based on target gene half-life and cell doubling time.

Materials: AUMsilence sdASO

Note: Activated T cells often show enhanced knockdown (70-90%) due to increased metabolic activity

Day 0

Functional Assays

At 48-72h post-ASO treatment, perform functional assays: cytokine ELISA (IFN-γ, IL-2, TNF-α), proliferation (CFSE dilution), cytotoxicity assays.

Materials: ELISA kits, flow cytometry antibodies

Note: AUMsilence preserves T cell function; no impact on proliferation or cytokine production in non-target pathways

Days 2-3

Jurkat Cells (Human T Cell Line)

Immortalized T cell line for high-throughput screening

Cell Culture

Culture Jurkat cells in RPMI-1640 + 10% FBS. Maintain log-phase growth (3-8 × 10⁵ cells/mL). Split cells to 3 × 10⁵ cells/mL 24h before ASO treatment.

Materials: Complete RPMI-1640

Note: Jurkat cells double every 20-24 hours

Day -1

AUMsilence Treatment

Add AUMsilence sdASO at 10 μM to Jurkat cultures (5 μM may be sufficient for some targets as immortalized lines often require lower concentrations).

Materials: AUMsilence sdASO

Note: Expect 65-85% knockdown in Jurkat cells at 48h

Day 0

Analysis

Harvest at 48h for qRT-PCR and flow cytometry. Jurkat cells useful for TCR signaling studies (PLC-γ1, ZAP70, LAT knockdown).

Materials: Standard molecular biology reagents

Note: Immortalized lines show less donor variability than primary cells

Day 2

Essential Controls

Untreated T Cells: Baseline expression levels and viability

Culture identically but without ASO addition

Non-Targeting Control ASO: Control for off-target effects and ASO-related toxicity

Use AUM non-targeting control at same concentration (10 μM or match experimental concentration)

Positive Control (Housekeeping Gene): Verify ASO uptake and RNase H activity

Optional: Use GAPDH or ACTB-targeting ASO to confirm 70-90% knockdown

Optimization Strategies

ASO Concentration

Recommendation: Start at 10 μM as the standard concentration. Use 5 μM for sensitive targets or higher concentrations for highly stable genes.

Rationale: Optimal concentration typically ranges from 1-20 μM depending on target gene half-life and cell doubling time. Rapidly dividing cells or highly stable transcripts may require higher concentrations.

Incubation Time

Recommendation: 48-72h for most applications. 24h for early time-point analysis.

Rationale: Gymnotic uptake completes by 12-24h. mRNA degradation peaks at 48-72h. Protein knockdown may require 72-96h for long-lived proteins.

Cell Density

Recommendation: Maintain 0.5-1.5 × 10⁶ cells/mL.

Rationale: T cells compete for ASO at high density. Very low density (<3 × 10⁵/mL) may reduce viability.

Medium Composition

Recommendation: Standard RPMI + 10% FBS is optimal.

Rationale: Serum proteins do not significantly inhibit gymnotic ASO uptake (unlike lipofection).

ASO Sequence Selection

Recommendation: Design and test 3-5 ASOs targeting different regions of the target mRNA. Select the sequence with highest knockdown efficiency for downstream experiments.

Rationale: Knockdown efficiency varies dramatically (30-90%) depending on target site due to RNA secondary structure, protein binding, and accessibility. Testing multiple sequences ensures you identify the optimal ASO. Target regions with low predicted secondary structure (ΔG > -10 kcal/mol) and avoid highly structured domains or protein binding sites when possible.

Troubleshooting

Low Knockdown Efficiency (<50%)

Cell Death or Toxicity (Viability <85%)

High Variability Between Donors

Protein Not Reduced Despite mRNA Knockdown

Validation Methods for T Cell Knockdown

Comprehensive validation ensures robust, reproducible results. AUMsilence preserves T cell viability for all downstream assays.

Quantitative RT-PCR (qRT-PCR)

Purpose: Gold standard for mRNA knockdown quantification

Protocol: Extract total RNA at 48-72h post-treatment. Use TaqMan or SYBR Green qPCR. Normalize to housekeeping genes (GAPDH, ACTB, HPRT1).

Expected Results: 70-95% mRNA reduction vs. untreated or non-targeting control

Tips: Include RNA quality check (260/280 ratio >1.8). Use biological triplicates for statistical power.

Flow Cytometry

Purpose: Validate surface marker or intracellular protein knockdown at single-cell resolution

Protocol: For surface proteins: Stain live cells with fluorescent antibodies at 72-96h. For intracellular proteins: Fix, permeabilize, and stain. Include viability dye (7-AAD, Live/Dead).

Expected Results: 50-85% protein reduction (MFI shift). >95% viability.

Tips: Use isotype controls. Gate on viable, singlet lymphocytes. Compare MFI between treated and control populations.

Western Blot

Purpose: Quantify total protein knockdown in bulk population

Protocol: Lyse cells at 72-96h post-treatment. Run SDS-PAGE and transfer. Probe with target-specific antibody. Normalize to loading control (β-actin, GAPDH, vinculin).

Expected Results: 60-85% protein reduction vs. control

Tips: Protein half-life affects timing. Long-lived proteins (>48h half-life) may require 96-120h for maximal knockdown.

ELISA (Cytokine Secretion)

Functional Assays

Purpose: Validate phenotypic consequences of gene knockdown

Functional Assays:

Proliferation (CFSE Dilution)

Protocol: Label T cells with CFSE before AUMsilence treatment. Stimulate with anti-CD3/CD28. Analyze CFSE dilution by flow cytometry at 72-96h.

Interpretation: Reduced proliferation if targeting pro-proliferative genes (IL-2, CD28, mTOR pathway).

Cytotoxicity Assay

Protocol: Co-culture knockdown T cells with target cells (tumor lines or peptide-loaded cells). Measure target cell lysis by LDH release, CFSE dilution, or flow-based viability.

Interpretation: Reduced killing if targeting perforin, granzyme B, or FasL. Enhanced killing if targeting checkpoints (PD-1, CTLA-4).

Apoptosis (Annexin V / 7-AAD)

Protocol: Stain with Annexin V and 7-AAD at 48-96h. Analyze by flow cytometry.

Interpretation: Increased apoptosis if targeting pro-survival genes (BCL2, MCL1). Decreased apoptosis if targeting Fas or FasL.

Activation Markers (CD25, CD69, CD71)

Protocol: Stimulate T cells post-knockdown. Measure CD25, CD69, CD71 expression by flow cytometry.

Interpretation: Reduced activation if targeting TCR signaling or co-stimulatory molecules.

Critical Controls for Validation

Untreated T Cells

Purpose: Baseline for all measurements

Culture identically to treated cells without ASO addition.

Non-Targeting Control ASO

Purpose: Control for ASO-specific effects (off-target, immune stimulation)

Use AUM non-targeting control ASO at same concentration and timing as experimental ASO.

Positive Control ASO

Purpose: Verify ASO uptake and activity

Optional but recommended: Use GAPDH or ACTB-targeting ASO to confirm 70-90% knockdown.

Viability Control

Purpose: Ensure cell health throughout experiment

Include viability staining (7-AAD, Live/Dead) in all flow cytometry panels. Expect >90% viability.

Dose-Response Verification

Purpose: Confirm concentration-dependent knockdown and rule out saturation effects

Test at minimum 3 concentrations (e.g., 5 μM, 10 μM, 20 μM AUMsilence). Knockdown should correlate with concentration. Absence of dose-dependency suggests off-target or non-specific effects. Essential for establishing optimal working concentration. Note: Optimal concentration typically ranges from 1-20 μM based on cell doubling time and target gene stability.

Independent ASO Verification

Purpose: Confirm target specificity with second independent ASO sequence

Use 3-5 different ASOs targeting non-overlapping regions of the same mRNA. Concordant knockdown across independent sequences confirms on-target specificity and eliminates sequence-specific off-target effects. This is the gold standard for definitive target validation.

Best Practices

Use biological triplicates (n=3 independent experiments) for statistical analysis
Validate knockdown at both mRNA (qRT-PCR) and protein (flow cytometry or Western blot) levels
Include time-course experiments for targets with unknown mRNA/protein half-lives
For functional assays, verify knockdown in the same cells used for functional readout
Report both knockdown efficiency and cell viability in all publications
Use appropriate statistical tests (t-test, ANOVA) with p<0.05 threshold

Research Applications in T Cell Biology

AUMsilence enables diverse applications across basic immunology, translational research, and therapeutic development.

Cancer Immunotherapy & CAR-T Engineering (4 applications)

Optimize adoptive cell therapies by silencing inhibitory pathways and enhancing T cell function

CAR-T Cell Enhancement

Approach: Silence PD-1, CTLA-4, or LAG-3 to prevent exhaustion in CAR-T cells. Combine with TGFβR2 knockdown for solid tumor resistance.

AUM Advantage: Transfection-free delivery preserves CAR-T viability and function. Compatible with clinical manufacturing workflows.

Tumor-Infiltrating Lymphocyte (TIL) Optimization

Approach: Enhance TIL cytotoxicity by silencing checkpoint receptors or TOX (exhaustion driver). Boost cytokine production (IFN-γ, TNF-α).

AUM Advantage: Works in ex vivo expanded TILs without altering activation state or proliferative capacity.

Cytokine Release Syndrome (CRS) Prevention

Approach: Knockdown TNF-α or IL-6 in CAR-T cells to reduce systemic inflammation while maintaining anti-tumor efficacy.

AUM Advantage: Selective cytokine silencing without broad immunosuppression.

Fratricide Prevention

Approach: Silence target antigen (e.g., CD7, CD5) in CAR-T cells or knockdown FasL to prevent CAR-T self-killing.

AUM Advantage: Enables targeting of T cell-expressed antigens in T cell malignancies.

T Cell Exhaustion & Dysfunction (3 applications)

Study and reverse T cell exhaustion in chronic infections and cancer

Checkpoint Receptor Studies

Approach: Systematically knock down PD-1, CTLA-4, LAG-3, TIM-3, TIGIT to define exhaustion hierarchy and functional rescue potential.

AUM Advantage: Multiplex knockdowns in same culture. No phenotype alteration from delivery method.

Exhaustion Transcription Factor Dissection

Approach: Target TOX, NR4A1, or NFAT to prevent exhaustion programming. Compare with checkpoint receptor silencing.

AUM Advantage: High efficiency in exhausted T cells despite low metabolic activity.

Functional Restoration Assays

Approach: Measure proliferation, cytokine production, and cytotoxicity after exhaustion driver knockdown. Compare to checkpoint blockade antibodies.

AUM Advantage: Preserved T cell function enables accurate functional readouts.

T Cell Differentiation & Polarization (3 applications)

Study lineage commitment and plasticity in CD4+ T helper subsets

Master Transcription Factor Studies

Approach: Knockdown T-bet (Th1), GATA3 (Th2), RORγt (Th17), or FOXP3 (Treg) to study lineage commitment and stability.

AUM Advantage: Works during differentiation without interfering with polarizing signals.

Cytokine Circuit Dissection

Approach: Silence cytokines (IL-2, IL-4, IL-10, IFN-γ) or cytokine receptors to define autocrine/paracrine feedback loops.

AUM Advantage: Knockdown cytokines without affecting differentiation-inducing factors in media.

Treg Suppression Mechanism Studies

Approach: Knockdown FOXP3, IL-10, TGF-β, or CTLA-4 in Tregs to dissect suppressive mechanisms. Co-culture with effector T cells.

AUM Advantage: High efficiency in Tregs, which are particularly resistant to transfection.

TCR Signaling & Activation (3 applications)

Dissect T cell receptor signaling cascades and activation pathways

Proximal TCR Signaling

Approach: Target LCK, ZAP70, LAT, SLP-76, or PLCγ1 in Jurkat cells or primary T cells. Measure calcium flux and ERK activation.

AUM Advantage: No electroporation-induced calcium perturbation. Clean functional readouts.

Co-stimulation Studies

Approach: Silence CD28, ICOS, or 4-1BB to define co-stimulatory requirements for activation, proliferation, and cytokine production.

AUM Advantage: Preserves surface receptor expression until knockdown achieved (unlike blocking antibodies).

Negative Regulators

Approach: Knockdown PTPN6 (SHP-1), PTPN11 (SHP-2), or UBASH3A to enhance TCR signaling strength.

AUM Advantage: Study intrinsic negative regulation without pathway agonists.

Autoimmune Disease Models (3 applications)

Model T cell contributions to autoimmunity and test therapeutic targets

Th17-Mediated Autoimmunity

Approach: Silence RORγt or IL-17A in differentiated Th17 cells. Test in co-culture models or adoptive transfer experiments.

AUM Advantage: High knockdown in Th17 cells enables functional suppression of pathogenic phenotype.

Regulatory T Cell Biology

Approach: Modulate FOXP3, IL-10, TGF-β, or CTLA-4 in Tregs to study suppressive mechanisms and stability.

AUM Advantage: Tregs are highly resistant to conventional transfection; AUMsilence achieves 60-85% knockdown.

Autoreactive T Cell Studies

Approach: Silence activation pathways or costimulatory molecules to prevent autoreactive T cell responses.

AUM Advantage: Enables mechanistic studies without systemic immunosuppression.

Basic T Cell Biology (3 applications)

Fundamental studies of T cell development, homeostasis, and function

Apoptosis Pathway Dissection

Approach: Target FAS, FASL, BCL2, BCL-XL, or BIM to define AICD mechanisms and survival requirements.

AUM Advantage: No lipofection-induced AICD confounding apoptosis studies.

Metabolic Reprogramming

Approach: Silence glycolysis (HK2, PFKFB3), oxidative phosphorylation (mitochondrial genes), or mTOR pathway components.

AUM Advantage: Study metabolism without transfection-induced metabolic stress.

MicroRNA Function

Approach: Use AUMantagomir to inhibit miR-155, miR-146a, miR-21, or miR-17-92 cluster. Study effects on differentiation and function.

AUM Advantage: Efficient microRNA inhibition in primary T cells.

Frequently Asked Questions

Why don't traditional transfection reagents work well in T cells?

T cells have low endocytic activity and are highly sensitive to lipid-based reagents. Lipofection triggers activation-induced cell death (AICD) through the Fas/FasL pathway, resulting in 40-60% cell death. Additionally, transfection efficiency is limited to a narrow 24-48h window post-activation. AUMsilence bypasses these issues through gymnotic delivery, which doesn't require endocytosis or cause membrane stress.

How does AUMsilence self-delivering ASO work without transfection?

Can I use AUMsilence in both resting and activated T cells?

Will AUMsilence treatment alter my T cell phenotype or function?

How long does knockdown last?

Is AUMsilence compatible with CAR-T manufacturing workflows?

Can I knock down multiple genes simultaneously?

What concentration should I use?

Start with 10 μM as the standard concentration for most applications. This concentration achieves optimal knockdown (70-95%) with minimal off-target effects. Use 5 μM for sensitive targets or cells with slower doubling times. Highly stable genes or rapidly dividing cells may require higher concentrations. Optimal concentration typically ranges from 1-20 μM depending on target gene half-life and cell doubling time.

How is this different from siRNA?

Do I need to optimize the protocol for different T cell subsets?

How do I validate knockdown?

Is there a risk of off-target effects?

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