Polysome Profiling Service
Tired of mismatches between mRNA and protein levels? Uncover hidden regulatory mechanisms with our advanced polysome profiling Service. By isolating ribosome-bound mRNAs via sucrose gradient ultracentrifugation and integrating multi-omics analysis, we quantify translational efficiency, spotlight regulatory bottlenecks, and reveal biomarkers.
Overview of Polysome Profiling
Polysome profiling is a powerful biochemical technique used to isolate and analyze messenger RNAs (mRNAs) based on the number of ribosomes they are associated with. This association directly reflects the translational activity of an mRNA. By separating mRNAs bound to multiple ribosomes (polysomes) from those bound to single ribosomes (monosomes) or no ribosomes, researchers can gain a snapshot of the "translatome"–the entirety of actively translated mRNAs within a cell at a given moment. This method provides crucial insights into gene expression regulation at the translational level, which often complements and extends findings from transcriptomic studies.
Fig.1 Polysome profiling followed by RNA-seq during cardiomyogenic differentiation.1
Sample Guidelines
To guarantee optimal results, kindly follow the sample requirements outlined below:
Adherent cells: ≥1×10⁷ cells (harvested at 80–90% confluency).
Suspension cells: ≥1×10⁷ cells (viability >90% recommended).
Yeast: ≥1×10⁸ cells (log-phase growth preferred).
Tissue samples: ≥100 mg (snap-frozen in liquid nitrogen).
Samples must be preserved in RNA-stable reagents or frozen immediately at -80°C. Ship on dry ice with insulated packaging to prevent thawing. For sample preparation details beyond these specifications (e.g., specialized lysis buffers, treatment protocols, or rare sample types), contact our technical team for tailored guidance before submission.
Experimental Workflow
Our polysome profiling workflow is meticulously designed to ensure high-quality data and actionable insights. Key steps involved:
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Consultation and Experimental Design
We begin with a thorough discussion of your research goals to optimize the polysome profiling strategy, including sample preparation protocols and downstream analysis choices. -
Sample Preparation and QC
Upon receipt, samples undergo quality assessment. Cells or tissues are gently lysed under conditions that preserve polysome integrity.
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Polysome Fractionation
Lysates are loaded onto sucrose density gradients and then ultracentrifuged. Gradients are then fractionated while continuously monitoring UV absorbance to identify non-translating (free mRNA, ribosomal subunits, monosomes) and translating (polysomes) fractions.
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RNA Isolation from Fractions
High-quality RNA is extracted from the relevant pooled fractions (e.g., non-translating, light polysomes, heavy polysomes) or individual fractions as per the experimental design. RNA quality and quantity are assessed.
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Downstream Analysis
Isolated RNA is typically subjected to library preparation and NGS to identify and quantify the mRNAs in each fraction. Alternatively, RT-qPCR can be used for targeted gene analysis.
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Bioinformatic AnalysisComprehensive data analysis is performed, including read alignment, transcript quantification, calculation of translational efficiency scores, differential translation analysis, and functional enrichment analysis.
Final Deliverables
1. Comprehensive Project Report
Including detailed methodology, raw and processed data (e.g., polysome profiles, sequencing reads), bioinformatic analysis results, and interpretation of findings.
2. High-Resolution Figures
Publication-quality visuals summarizing key results, such as polysome profile traces, heatmaps of differentially translated genes, and gene set enrichment plots.
3. Processed Data Files
Including gene expression tables, translational efficiency scores, and lists of significantly altered genes.
Case Study
This case study employed polysome profiling to analyze and compare ribosomal components across distinct experimental conditions, unveiling crucial insights into translational dynamics.
 Fig.2 Ribosome fraction separation diagram. |
 Fig.3 Comparison of translation efficiency. |
 Fig.4 Differentially expressed analysis. |
 Fig.5 Correlation analysis. |
 Fig.6 GO enrichment. |
 Fig.7 KEGG enrichment bubble chart. |
Applications
Our polysome profiling service offers critical insights across a diverse range of research fields by directly measuring the translational status of genes. Key applications include:
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Drug Discovery and Development
- Identifying molecular targets of novel therapeutic compounds.
- Understanding mechanisms of drug action and resistance by observing changes in the translation of specific mRNAs.
- Assessing the translational impact of drug candidates on cellular pathways.
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Cancer Biology
- Discovering dysregulated translational pathways in cancer cells.
- Identifying mRNAs whose translation is specifically altered during tumor progression and metastasis.
- Uncovering novel biomarkers for cancer diagnosis, prognosis, or therapeutic response.
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Disease Mechanism Elucidation
- Investigating how translational control contributes to the pathology of various diseases.
- Identifying specific proteins whose synthesis rates are altered in disease states.
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Developmental Biology
- Investigating how translational regulation governs cell fate determination, differentiation, and morphogenesis during embryonic development.
- Analyzing changes in protein synthesis patterns during different developmental stages.
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Basic Research in Gene Regulation
- Uncovering fundamental mechanisms of translational control, including the roles of microRNAs, RNA-binding proteins, and upstream open reading frames (uORFs).
- Validating targets of translational regulators.
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Neuroscience
- Exploring the role of local protein synthesis in neuronal function, synaptic plasticity, and memory formation.
- Identifying translationally regulated mRNAs in specific brain regions or neuronal subtypes.
Why Choose Creative Biolabs?
- Expert Translational Solutions
Partner with specialists who bridge transcriptomics and proteomics. We design experiments to capture active translation, ensuring your data reflects functional gene expression, not just mRNA levels.
- Cutting-Edge Technology
Access state-of-the-art polysome isolation and sequencing (Polysome-Seq). Paired with optimized protocols, we deliver robust data across diverse samples, from human tissues to microbial cultures.
- End-to-End Scientific Support
From experimental design to advanced bioinformatics, our team provides seamless guidance. We decode translational efficiency metrics and regulatory mechanisms, aligning findings with your research goals.
- Reproducible Data
Benefit from rigorous QC and standardized workflows. Our data directly answers your biological questions, backed by a proven track record in high-impact journals.
FAQs
Q: What sucrose gradient and fractionation strategy are used in polysome profiling experiments?
A: Our standard protocol utilizes a 10%-45% sucrose density gradient for ultracentrifugation, yielding 18 fractions to capture monosomes, polysomes, and free mRNA subpopulations. This approach ensures high-resolution separation of ribosome complexes. For projects requiring specialized optimization, such as gradient range adjustments or fraction consolidation, we provide fully customizable workflows aligned with your specific biological targets.
Q: What samples work for polysome profiling?
A: Our service works with various samples, like cultured cells, tissues from different organisms, yeast, and bacteria. The main thing is proper collection and preservation to keep polysomes intact. We offer tailored sample-prep guidance.
Q: What are the key benefits of using polysome profiling for studying gene regulation?
A: Its key advantage is directly assessing translational efficiency. It helps identify genes regulated at the protein-synthesis level, uncover translational control mechanisms, and understand how cells adjust protein production quickly in response to stimuli without changing mRNA levels.
Q: How long does the polysome profiling Service typically take from start to finish?
A: From sample receipt to delivery of your final report, the service usually takes 6 to 10 weeks. However, the exact timeline may vary based on factors such as the number of samples, the complexity of your experimental design, the depth of sequencing chosen, and any specialized bioinformatics analyses required. We will provide a detailed project timeline upfront and keep you updated as your study progresses.
Q: How does polysome profiling differ from standard RNA-Seq?
A: Standard RNA-Seq measures total mRNA abundance, which may not match protein levels. Polysome profiling, especially with sequencing (Polysome-Seq), quantifies mRNAs actively being translated. This gives a more direct measure of gene expression at the protein-synthesis level, highlighting key regulatory steps transcriptomics misses. We can advise on the best approach or combo for your research.
Related Services
- Ribosome profiling
For high-resolution mapping of ribosome positions on mRNA, providing detailed insights into translation initiation sites, elongation dynamics, and novel ORFs.
- RNA-Seq Services
Comprehensive transcriptome analysis, including mRNA, total RNA, small RNA, and long non-coding RNA sequencing, to correlate translational data with overall transcript abundance.
- Quantitative Proteomics Services
Directly measure protein abundance using mass spectrometry-based approaches to validate and complement findings from translatome studies.
Creative Biolabs is committed to providing cutting-edge polysome profiling services that empower researchers to unravel the complexities of translational regulation. Discover how we can accelerate your research, request a consultation today.
Reference
- Pereira, Isabela Tiemy, et al. "Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs." Scientific Data 5.1 (2018): 1-11. Distributed under Open Access license CC BY 4.0, without modification.
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