Translating Ribosome Affinity Purification (TRAP) Service
Are you grappling with challenges in dissecting gene expression within specific cell types in complex tissues, struggling to distinguish transcriptional shifts from actual protein synthesis, or finding bulk RNA analysis inadequate for your nuanced research questions? Our advanced translating ribosome affinity purification (TRAP) service helps you capture a precise snapshot of actively translating mRNA directly from your target cell populations in their native environment. This is achieved through state-of-the-art techniques involving cell-type-specific expression of epitope-tagged ribosomal proteins and highly efficient immunopurification, followed by next-generation sequencing.
Overview of TRAP
Understanding gene expression in complex, multicellular organisms requires dissecting molecular events within specific cell types. While transcriptomic analyses provide valuable information, the abundance of mRNA does not always directly correlate with protein synthesis rates due to extensive translational control. TRAP is a powerful technique developed to address this by enabling the isolation and analysis of actively translating mRNAs from genetically defined cell populations within intact tissues. It typically involves cell-specific expression of an epitope-tagged ribosomal protein, allowing for affinity purification of polysomes and their associated mRNAs, providing a precise snapshot of ongoing protein synthesis.
Fig.1 Polysome profiling using translating ribosome affinity purification (TRAP).1
Streamlined TRAP Workflow
Our TRAP service follows a meticulously designed workflow to ensure the highest quality data and actionable insights. This process is transparent and collaborative, keeping you informed at every stage. Key Steps Involved:
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Initial Consultation and Experimental Design
We begin with an in-depth discussion to understand your project goals, specific cell types of interest, characteristics of your model system, and desired outcomes. Together, we will finalize a tailored experimental plan, including controls and replicates. -
Sample Quality Control and Lysate Preparation
Upon receipt, samples undergo rigorous quality assessment. Tissues or cells are then gently lysed under conditions optimized to maintain polysome integrity and prevent RNA degradation.
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Affinity Purification of Tagged Ribosomes
The core of the TRAP technique involves the highly specific immunopurification of the epitope-tagged ribosomes from the cell lysate using high-affinity antibodies coupled to magnetic beads or an equivalent matrix.
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RNA Isolation and Quality Assessment
mRNA is carefully extracted from the purified polysomes. The yield and integrity of the isolated RNA are meticulously assessed to ensure suitability for downstream applications.
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NGS Library Preparation
High-quality cDNA libraries are constructed from the TRAP-isolated mRNA using protocols optimized for low-input samples.
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Sequencing and Bioinformatic AnalysisLibraries are sequenced on advanced NGS platforms to generate high-depth transcriptomic data. Our bioinformatics pipeline includes raw data quality control, read alignment to the relevant genome, quantification of gene expression, differential expression analysis between conditions/cell types, pathway, and gene ontology enrichment analysis, and visualization of results.
Main Applications
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Neuroscience
- Dissecting the molecular profiles of distinct neuronal and glial cell populations in different brain regions.
- Identifying cell-type-specific translational responses to stimuli, learning, memory formation, or neurological disease progression.
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Oncology
- Characterizing the translatomes of cancer cells versus stromal cells within the tumor microenvironment.
- Identifying translational reprogramming in response to drug treatments, aiding in understanding mechanisms of action and resistance.
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Immunology
- Analyzing the translational status of specific immune cell subsets during an immune response, infection, or autoimmune disease.
- Understanding how different immune cells alter their protein synthesis machinery upon activation or differentiation.
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Developmental Biology
- Tracking changes in protein synthesis in specific cell lineages during embryonic development and tissue differentiation.
- Identifying key translational control points that govern cell fate decisions and morphogenesis.
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Drug Discovery and Development
- Validating drug targets by confirming their active translation in relevant cell types.
- Assessing the cell-type-specific efficacy and off-target effects of novel therapeutic compounds by monitoring changes in the translatome.
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Metabolic Research
- Investigating how different cell types within metabolic organs adapt their protein synthesis in response to dietary changes or metabolic stress.
- Identifying cell-specific translational signatures associated with metabolic disorders like diabetes or obesity.
Core Advantages
- Deep Scientific Expertise
Our team comprises seasoned molecular biologists and bioinformaticians with extensive experience in TRAP methodologies, RNA biology, and next-generation sequencing. We have successfully applied TRAP to diverse research areas, including challenging tissues like the brain and the analysis of rare cell populations.
- Optimized Protocols
We employ meticulously optimized protocols for tissue lysis, polysome immunopurification, RNA extraction, and library preparation to ensure maximal yield and quality of translatomic RNA, even from limited starting material.
- Cell-Type Specificity
Our approach ensures genuine cell-type-specific translatome profiling, avoiding the confounding factors of tissue heterogeneity and the potential artifacts introduced by cell dissociation methods.
- Comprehensive Bioinformatic Analysis
We provide more than just raw data. Our bioinformaticians deliver in-depth analysis, translating complex datasets into meaningful biological insights, complete with publication-ready figures and reports.
- Focus on Translational Control
Unlike standard transcriptomic analyses that measure total mRNA, TRAP focuses on actively translating mRNAs, offering a more direct and dynamic view of protein synthesis and cellular responses. Published Data consistently show that changes in the translatome often occur independently of changes in the transcriptome.
- Collaborative Approach
We believe in working closely with our clients, offering expert consultation from experimental design through to data interpretation and publication support.
FAQs
Q: What are the main advantages of TRAP-Seq compared to single-cell RNA-Seq (scRNA-Seq) for my research?
A: TRAP-Seq captures actively translating mRNAs from your target cells in their native tissue, giving a clearer picture of protein synthesis. While scRNA-Seq provides transcriptome data at single-cell resolution, TRAP-Seq is often better for assessing the translatome without the artifacts that can arise from the enzymatic dissociation needed for scRNA-Seq.
Q: How much starting material is typically required for a TRAP experiment?
A: One of TRAP's advantages is that it usually needs less starting material than older bulk methods, like traditional polysome profiling, especially when targeting specific cell types. The exact amount depends on factors like the abundance of your target cells in the tissue, the size of the tissue sample, and the expression level of the tagged ribosome.
Q: What bioinformatics support does Creative Biolabs offer for TRAP-Seq data?
A: Creative Biolabs offers full bioinformatics support for your TRAP-Seq data, from initial quality control and adapter trimming to alignment with the reference genome, quantification of gene expression at the transcriptome level, differential gene expression analysis between groups, and functional enrichment analyses.
Creative Biolabs is dedicated to providing cutting-edge TRAP services that deliver precise, cell-specific insights into translational regulation. Ready to explore how our TRAP service can illuminate the translatome in your cells of interest? Our expert team is available to discuss your project needs.
Reference
- Goldenkova-Pavlova, Irina V., et al. "Computational and experimental tools to monitor the changes in translation efficiency of plant mRNA on a genome-wide scale: advantages, limitations, and solutions." International Journal of Molecular Sciences 20.1 (2018): 33. Distributed under Open Access license CC BY 4.0, without modification.
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