Ribosome Profiling Service
Our ribosome profiling service offers an unparalleled view into the dynamic world of protein synthesis. Are you grappling with understanding true gene expression beyond simple mRNA abundance, identifying novel drug targets missed by transcriptomics alone, or deciphering complex translational regulatory networks? Our ribosome profiling service helps you gain unprecedented insights into active protein synthesis and translational control through advanced next-generation sequencing of ribosome-protected mRNA fragments and sophisticated, customized bioinformatics analysis, empowering your research development.
Introduction of Ribosome Profiling
Ribosome profiling, or Ribo-seq, is a powerful technique that provides a "snapshot" of all ribosomes actively translating mRNA in a cell at a specific moment. By sequencing the mRNA fragments protected by these ribosomes, researchers can quantitatively measure gene expression at the translational level, identify the exact regions of mRNA being translated (including novel ORFs), and assess the efficiency of protein synthesis on a genome-wide scale. This method offers deeper insights than transcriptomics alone, revealing crucial regulatory mechanisms that control protein production and cellular function.
Fig.1 Schematic illustration of the current workflow of ribosomes.1
Sample Requirements
Sample Delivery Requirements
For best results, please prepare and ship your samples carefully. Always discuss specific needs with our team before sending. A completed sample submission form detailing your experiment must be included with every shipment.
1. Cell Pellets
Amount: Generally, ≥1×10⁷ cells per sample. Consult us, as this can vary.
Prep: Harvest quickly. Wash with ice-cold PBS. Flash-freeze in liquid nitrogen. Store at -80°C.
2. Tissue Samples
Amount: Around 100 mg per sample, depending on tissue type.
Preferred: Rapidly excise and immediately flash-freeze in liquid nitrogen.
3. Yeast/Bacteria Cultures
Amount: Sufficient culture for required cell mass (discuss with us).
Preferred: Harvest rapidly. If using translation inhibitors (e.g., cycloheximide), add them shortly before harvest. Wash pellets with ice-cold buffer. Flash-freeze in liquid nitrogen.
Workflow
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Initial Consultation & Experimental Design
A thorough discussion of your research objectives, experimental setup, sample types, and desired outcomes. Our experts provide guidance on optimal sample preparation and number of replicates. -
Sample Quality Control (QC) & Lysate Preparation
Upon sample receipt, we perform rigorous QC checks. Cells or tissues are lysed under conditions that preserve the integrity of ribosome-mRNA complexes, often involving rapid inhibition of translation elongation.
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Ribosome Footprint Generation & Purification
Lysates are treated with RNases to digest mRNA regions not protected by ribosomes. The resulting ~28-30 nucleotide mRNA fragments bound by ribosomes are then isolated, typically through sucrose density gradient centrifugation or size-exclusion chromatography.
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Library Construction for Sequencing
The isolated RPFs are converted into a cDNA library suitable for sequencing. This involves RNA fragmentation, adapter ligation to both 3' and 5' ends, reverse transcription, PCR amplification, and stringent size selection of the final library.
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Next-Generation Sequencing (NGS)
The prepared libraries are sequenced on high-throughput Illumina platforms, generating millions of short reads per sample.
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Bioinformatics Analysis & Data InterpretationComprehensive bioinformatics pipeline including raw read QC, adapter trimming, removal of rRNA contaminants, alignment of RPF reads to the reference genome/transcriptome, quantification of ribosome occupancy per gene/transcript, analysis of P-site offsets, identification of translated ORFs, and differential translation analysis.
Final Deliverables
1. Detailed Project Report: Summarizing the methodology, quality control metrics for each step, key findings with graphical representations, and interpretation of the results in the context of your project.
2. Raw Sequencing Data: FASTQ files for each sample.
3. Processed Data Files: Including aligned reads, read count tables for genes/transcripts, lists of identified translated ORFs, differential translation analysis results, and any custom analyses performed.
Case Study
This case study employed ribosome profiling to examine translational dynamics across various cellular conditions, aiming to unravel critical insights into gene expression regulation at the ribosomal level.
 Fig.2 Reads base composition distribution. |
 Fig.3 Distribution of reading frames. |
 Fig.4 Per sequence GC content. |
 Fig.5 Differential gene volcano diagram. |
 Fig.6 Differential GO enrichment histogram. |
 Fig.7 Gene KEGG enrichment bubble chart. |
 Fig.8 Pearson correlation analysis. |
 Fig.9 Protein network interaction. |
 Fig.10 ORF classification. |
Core Applications
Our ribosome profiling Service provides critical data for a wide range of research applications, including but not limited to:
- Identifying Actively Translated Genes
Determine precisely which mRNAs are being translated into proteins under specific conditions, offering a more accurate reflection of gene expression than transcriptomics alone.
- Quantifying Translational Efficiency
Measure the rate of protein synthesis for thousands of genes simultaneously, revealing critical points of translational control.
- Discovering Novel Translational Events: Identify unannotated open reading frames (ORFs), including upstream ORFs (uORFs) and non-canonical start sites, expanding the known proteome.
- Elucidating Mechanisms of Action
Understand how your compounds or genetic modifications impact protein synthesis, providing critical insights for drug development and functional genomics.
- Pinpointing Drug Targets & Biomarkers
Uncover disease-specific translational signatures or identify novel therapeutic targets regulated at the level of protein synthesis.
- Understanding Basic Biology
Investigate fundamental aspects of gene regulation, ribosome function, and cellular responses to stimuli.
- Validating RNA-Seq Findings
Confirm whether changes in mRNA abundance translate to corresponding changes in protein synthesis.
Key Strengths
Choosing Creative Biolabs for your ribosome profiling needs means partnering with a team dedicated to scientific excellence and client success.
- Deep Expertise
Our scientists possess extensive experience in RNA biology, NGS technologies, and advanced bioinformatics, ensuring your project is in capable hands.
- Cutting-Edge Technology
We utilize state-of-the-art sequencing platforms and validated protocols to deliver high-quality, reproducible data.
- Customized Bioinformatics
We don't believe in one-size-fits-all. Our bioinformatics solutions are tailored to your specific research questions, providing deeper insights beyond standard analyses. This includes robust P-site determination, ORF calling, and translational efficiency calculations.
- Rigorous Quality Control
Stringent QC checkpoints are integrated throughout the workflow, from sample intake to final data delivery, guaranteeing reliable results.
- Collaborative Approach
We work as an extension of your team, offering pre-project consultation, regular updates, and post-project support to help you interpret and leverage your data effectively.
- Proven Track Record
Published data and client testimonials attest to the quality and impact of our services in advancing research across various biological disciplines.
FAQs
Q: How does ribosome profiling differ from standard RNA-Seq, and when should I choose it?
A: RNA-Seq measures the abundance of mRNA transcripts, indicating which genes are transcribed. Ribosome profiling, however, directly measures active translation by sequencing only those mRNA fragments actively occupied by ribosomes. Choose ribosome profiling when you need to understand post-transcriptional gene regulation, identify actively translated ORFs, assess translational efficiency, or study the mechanism of drugs affecting protein synthesis.
Q: What kind of biological questions can ribosome profiling help answer?
A: Ribosome profiling is versatile and can address numerous questions, such as: Which genes change their protein synthesis rates under stress or drug treatment? Are there alternative translation start sites being used? Are non-coding RNAs being translated? How does ribosome occupancy change during different cell cycle stages or developmental processes? It's invaluable for drug discovery, understanding disease mechanisms, and fundamental biology.
Q: Can ribosome profiling analyze translational efficiency, ribosome stalls, and frame shifting?
A: Yes! Ribosome profiling quantifies translational efficiency by comparing ribosome density to mRNA levels, detects ribosome stalls via localized RPF pileups, and identifies frameshifting by analyzing codon-by-codon triplet periodicity deviations.
At Creative Biolabs, our ribosome profiling service empowers researchers to delve deeper into the complexities of gene expression by providing a direct and quantitative measure of protein synthesis. From meticulous sample processing to sophisticated bioinformatics analysis and expert consultation, we are committed to delivering high-quality, actionable insights that accelerate your scientific discoveries.
Reference
- Bagheri, Atefeh, et al. "Tracing translational footprint by Ribo-Seq: principle, workflow, and applications to understand the mechanism of Human diseases." Cells 11.19 (2022): 2966. Distributed under Open Access license CC BY 4.0, without modification.
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