mRNA stability is a key bottleneck for mRNA therapeutics, with half-life determining therapeutic protein expression duration and magnitude. Our Actinomycin D-based Evaluation Service uses the gold-standard transcription inhibition assay to quantify mRNA decay kinetics.
Creative Biolabs delivers rigorous, reliable data to inform sequence modifications and delivery strategies. We accelerate lead optimization, de-risk clinical programs, and ensure your constructs meet cellular performance demands for successful translation.
Fig.1 Schematic diagram of the structural
formula of actinomycin D.1,3
Actinomycin D specifically inhibits gene transcription by intercalating into cellular DNA, preventing RNA polymerase binding, and completely terminating de novo mRNA synthesis. With no new mRNA replenished, the existing target mRNA in cells degrades naturally. By collecting cells at different time points, extracting RNA, and quantitatively detecting the remaining target mRNA levels, the degradation pattern over time can be tracked. Finally, nonlinear regression curve fitting is used to calculate the half-life (t1/2), with a longer half-life indicating stronger mRNA stability and vice versa.
| Indicator | Description | Detection Method/Platform |
|---|---|---|
| mRNA Half-Life (t1/2) | The time required for 50% of the initial mRNA population to decay after transcription arrest. It is the primary metric for mRNA stability. | Real-Time Quantitative PCR (RT-qPCR): Gold-standard method for quantifying remaining mRNA copies. Data is processed via Non-linear Regression curve fitting. |
| mRNA Decay Rate (k) | The first-order rate constant of degradation. It is mathematically derived from the t1/2 value (k = ln(2)/t1/2). | RT-qPCR and Advanced Kinetic Modeling (e.g., one-phase exponential decay fitting in specialized software). |
| Relative mRNA Abundance | The quantity of the target transcript was normalized to a stable reference (e.g., 18S rRNA) at defined time points. It is used to plot the decay curve. | RT-qPCR (ΔCt and 2⁻ΔΔCt calculations). |
Culture target cells under optimal conditions (custom in vitro models if needed). Treat with optimized Actinomycin D (Act D) to inhibit de novo transcription via DNA intercalation, initiating decay measurement.
Harvest cells at predefined time intervals (e.g., every 2 hours) post-Act D treatment. Rapid processing ensures accurate temporal data reflecting true decay kinetics.
Extract high-quality total RNA from each time point, with rigorous DNase treatment to eliminate genomic DNA contamination and avoid qPCR bias.
Reverse transcribe purified RNA into cDNA. Perform qPCR with target-specific primers to precisely measure remaining mRNA abundance across a broad dynamic range.
Normalize qPCR Ct values and apply advanced non-linear regression (one-phase decay) to accurately calculate mRNA half-life (t1/2) in hours.
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We offer customized solutions built on scientific excellence, transforming stability measurement from a simple readout into a strategic optimization tool for your mRNA program.
Custom mRNA Construct Vetting:
Tailored kinetic analysis for client-specified mRNA constructs (including modified nucleotides, novel capping strategies) to assess post-transfection stability.
Gold-Standard Act D Assay Mastery:
Implement universally accepted Actinomycin D transcription inhibition assay, optimized for reliable, non-exogenous measurement of true biological half-life (t1/2).
Disease Model Customization:
Run assays in client-provided or specialized cell lines (e.g., iPSCs, senescent cells) to predict mRNA performance in compromised/aged cellular environments.
Quantitative and Publishable Data:
Use optimized RT-qPCR protocols and rigorous non-linear regression modeling for statistically robust decay kinetics, suitable for publication or regulatory packages.
Actionable Optimization Intelligence:
Provide expert interpretation and concrete sequence modification recommendations (e.g., UTR, poly(A) tail length) to evade degradation pathways like NMD.
Seamless PK/PD Integration:
Link mRNA stability data to delivery and pharmacokinetics (PK) assessments via Creative Biolabs' comprehensive analytical platforms for a complete translational picture.
Typically, the stability of mRNA is evaluated by treating the target cells in the experimental group and the control group with 2 µg/ml actinomycin D and measuring the half-life. In the experiment shown in the figure below, cells were treated with actinomycin D (2 μg/mL) for 0, 2, 4, 6, 8, 10, and 12 hours. Total RNA was extracted and analyzed using RT-qPCR. The main markers analyzed were DGS2, COLEC10, HSD17B2, ATF3, etc.
Fig.2 Cells were treated with actinomycin D, total RNA was extracted, and analyzed using RT-qPCR.2,3
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Act D is cytotoxic at high concentrations. We rigorously optimize its concentration and exposure time to fully inhibit transcription without causing non-specific cellular distress that alters mRNA decay, using multiple replicates to maintain measurement integrity.
Yes. By testing unencapsulated mRNA in basic cell models and comparing with LNP-delivered mRNA in relevant cell types, we precisely identify if instability stems from intrinsic sequence features or delivery-related issues (e.g., endosomal escape).
The Act D assay measures endogenous/natively introduced mRNA decay without complex reporter vectors, offering rapid foundational lead characterization. Inducible promoters avoid cytotoxicity but are more time-consuming to establish.
Turnaround is 3–6 weeks, depending on cell model complexity and time points. Contact our dedicated project management team to discuss needs and timeline goals for immediate scoping and prioritization.
Creative Biolabs provides a full spectrum of high-end services for mRNA therapeutic development, with the Actinomycin D based Evaluation Service serving as the core foundation for robust sequence characterization and optimization. Our commitment to using rigorous, published methodologies and providing kinetically precise data empowers your project to move confidently toward clinical milestones.
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