Advanced therapeutics like mRNA vaccines and gene editing tools have outpaced traditional analytical methods, with in vivo gene expression dynamics and heterogeneity posing core challenges. Creative Biolabs' PrimeFlow RNA Assay Service enables simultaneous single-cell quantification of RNA and protein expression. Leveraging proprietary bDNA signal amplification and advanced flow cytometry, it delivers high-resolution data on RNA dose, protein product, and cell functional state, resolving amplification effects and heterogeneity to support predictable dosing and clinical success.
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Creative Biolabs employs the core principles of proprietary branched-DNA (bDNA) technology combined with fluorescence in situ hybridization (FISH) to analyze millions of single cells by flow cytometry. This methodology allows for the physical co-localization and quantification of RNA transcripts and protein markers within the same cell, providing a complete picture of cellular function and therapeutic action.
The assay works by targeting the specific RNA of interest with a capture probe, followed by sequential hybridization and signal amplification using a branched-DNA structure. A single RNA molecule can anchor thousands of fluorescent reporter molecules, resulting in an exceptionally bright, highly specific signal that is easily resolved by standard flow cytometry instrumentation. This amplification overcomes the inherent low-abundance challenge of most functional RNA transcripts, delivering a sensitivity previously unattainable in flow-based applications.
Fig.1 Schematic diagram of the application of branched-strand DNA chemistry and SmartFlare chemistry during the PrimeFlow experiment.1
Creative Biolabs follows a meticulously structured, transparent process designed to integrate seamlessly with your R&D pipeline.
Define target RNA/protein markers, establish experimental controls, and select fluorochromes.
Design and proprietary synthesis of branched-DNA probe sets for unique targets.
Titrate probes and antibodies; establish optimal fixation, permeabilization, and hybridization protocols for the cell type.
Run the optimized protocol for RNA-FISH, protein immunolabeling, and flow cytometry acquisition.
Advanced bioinformatic processing to quantify co-localization and generate kinetic and heterogeneity metrics.
As biology experts, you require absolute confidence that your analytical data accurately reflects the complex in vivo reality of your therapeutic candidates. The Creative Biolabs PrimeFlow RNA Assay Service delivers this confidence by providing the highest level of resolution available in single-cell analysis.
Fully Custom Probe Design for Proprietary Targets:
No off-the-shelf solutions—custom bDNA probe sets for unique therapeutic mRNAs, gRNAs, lncRNAs, ensuring unmatched specificity for proprietary sequences.
One-Stop Transcriptional & Translational Validation:
Streamlined workflow from sample prep and specialized staining to bioinformatic kinetic modeling, delivering simultaneous RNA-Protein data to resolve translational disconnect.
Multiplexed Quality-by-Design (QbD) Analysis:
High-throughput screening of millions of single cells per run; acts as a Process Analytical Technology (PAT) tool to monitor delivery system efficiency and functional consistency.
Expert Protocol Optimization for Difficult Samples:
Specialists develop gentle, tailored protocols to preserve RNA/protein epitope integrity, even for complex samples (dissociated tumor tissues, rare cell subsets).
Unrivaled Sensitivity for Low-Abundance Transcripts:
Proprietary bDNA amplification enables robust detection of low-copy transcripts (1–10 copies per cell), providing gene expression evidence where standard methods fail.
High-Resolution Single-Cell Data:
Quantitative FCS file-based data supports statistical analysis and visualization (t-SNE, UMAP), enabling clear identification and quantification of distinct cell subsets in heterogeneous samples.
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To simultaneously detect mRNA, target proteins, and mitochondria, PrimeFlow technology can be adopted. First, the cells to be tested were stained with surface antibodies (CD4, CD44, OX40, CD71), and then co-incubated with MDR to detect mitochondrial function within the cells. Next, the PrimeFlow step is carried out. After one fixation, intracellular staining of Foxp3 and Ki67 is performed. After another fixation, annealing is conducted using target mRNA-specific oligonucleotide probes. Finally, a continuous hybridization step based on branched DNA chemistry is carried out, and detection is performed using a flow cytometer.
Fig.2 The expression of target markers and mRNA was simultaneously detected by flow cytometry and PrimeFlow technology.1
While qRT-PCR provides excellent sensitivity for bulk RNA quantification, it averages data across millions of cells, masking critical heterogeneity. Our service gives you the gene expression level per single cell and correlates it directly with protein expression. This is essential for therapeutics, where success depends on the functional status of a specific cell subset, not the average of a mixed population.
Yes, our proprietary bDNA amplification technology provides the necessary high sensitivity to robustly detect low-copy non-coding RNA targets (miRNA, lncRNA) and simultaneously phenotype the cell population in which they are expressed. We regularly design and validate custom probes for novel non-coding regulatory elements.
Our optimized protocol is compatible with a wide range of mammalian samples, including human whole blood, PBMCs, dissociated tissues (e.g., tumor biopsies), and established cell lines. Our team customizes the fixation and permeabilization steps to ensure maximal RNA and protein integrity for your unique sample type.
Our optimized protocol is significantly gentler than standard RNA-FISH methods. It is validated for simultaneous use with a wide array of surface and intracellular protein antibodies. We perform co-staining optimization as a key step in our workflow to ensure the epitopes for your required protein markers are preserved and detectable, maximizing the information gained from each sample.
Co-detection resolves the translational disconnect. An LNP might deliver 100% of its payload (RNA) to a cell, but only 20% of that RNA may translate into functional protein due to cellular constraints. Our assay allows you to quantify this precise gap, offering unparalleled insight into drug mechanism of action, translational efficiency, and ultimately, accurate dose optimization.
Creative Biolabs' PrimeFlow RNA Assay Service provides the vital single-cell analytical precision required to advance next-generation therapeutics. By combining proprietary bDNA amplification with high-parameter flow cytometry, we deliver the definitive data linking the dose (RNA) to the functional outcome (protein and phenotype). We empower you to resolve heterogeneity, optimize dosing, and accelerate your path to clinical success with confidence.
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