Identification of Circular RNAs using RNA Sequencing
November 14th, 2019
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Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.
Overview
This protocol details the preparation of circular RNA (circRNA) sequencing libraries, emphasizing the evaluation of various parameters for optimal circRNA detection. It provides insights into the impact of RNase R treatment and input amounts on circRNA identification.
Key Study Components
Area of Science
- Neuroscience
- RNA Biology
- Genomics
Background
- Circular RNAs are non-coding RNAs involved in transcriptional regulation.
- Understanding circRNA expression is crucial for elucidating their functional roles.
- Library preparation techniques can significantly affect circRNA detection.
- Proper handling of RNA samples is essential for successful experiments.
Purpose of Study
- To optimize circRNA library preparation protocols.
- To assess the effects of different parameters on circRNA detection.
- To facilitate the identification of circRNAs across various sample types.
Methods Used
- Stranded total RNA library preparation.
- RNase R pre-treatment to enrich circRNAs.
- Evaluation of kit types and input amounts.
- Assessment of RNA integrity using Agilent Bioanalyzer or TapeStation.
Main Results
- Optimal conditions for circRNA detection were established.
- Data on adjustable parameters were provided for user flexibility.
- Identifying circRNAs enhances understanding of their expression distribution.
- The method is applicable to any total RNA sample.
Conclusions
- This protocol aids in the effective preparation of circRNA sequencing libraries.
- It sets the groundwork for future studies on circRNA functions.
- Proper RNA handling and preparation are critical for successful outcomes.
Frequently Asked Questions
What are circular RNAs?
Circular RNAs are a type of non-coding RNA that may play roles in regulating gene expression and protein interactions.
Why is RNase R treatment important?
RNase R treatment is crucial for enriching circular RNAs by degrading linear RNAs, thus improving detection.
How can I assess RNA quality before library preparation?
RNA quality can be assessed using the RIN and DV200 values on an Agilent Bioanalyzer or TapeStation.
Can this method be used for any RNA sample?
Yes, this method is applicable to any total RNA sample.
What is the significance of identifying circRNAs?
Identifying circRNAs helps in understanding their expression patterns and potential functional roles in various biological processes.
Executive Industry Relevance
Circular RNA detection enables target validation in non-coding RNA biology, supporting mechanistic de-risking in early discovery. Optimized library preparation improves predictive confidence for circRNA biomarker identification across tissue types. This workflow enhances assay development for neurological and peripheral disease models where circRNA enrichment correlates with biological relevance.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of circRNA-mediated miRNA regulation and protein-protein interaction networks.
- Operational Value: Provides standardized workflow for functional target validation of non-coding RNA candidates.
- Scientific Value: Supports biological de-risking by clarifying circRNA expression patterns in disease-relevant systems.
Screening & Assay Development
- Scientific Value: Generates quantitative circRNA expression data for assay standardization and reproducibility.
- Operational Value: Enables scalable screening platforms using stranded total RNA with RNase R pretreatment.
- Scientific Value: Facilitates preparation of validated biological systems for downstream compound screening and target engagement studies.
Translational & Preclinical Research
- Scientific Value: Demonstrates disease-relevant system applicability through cross-tissue circRNA abundance profiling.
- Operational Value: Supports translational biomarker alignment by identifying tissue-specific circRNA signatures.
- Scientific Value: Enhances predictive confidence in preclinical models via conserved circRNA detection across human tissue types.
Pipeline & Workflow Integration
This method integrates into early discovery workflows to support hypothesis testing and pathway clarification prior to lead identification efforts.
- Discovery Biology: Supports mechanistic de-risking by enabling detection of circRNAs involved in transcriptional regulation and miRNA sponging.
- Screening: Delivers assay-ready, quantitative circRNA measurements for reproducible compound evaluation.
- Analytics: Provides normalized circRNA counts from multiple detection tools for comparative condition analysis.
- Translational Research: Connects discovery to preclinical continuity through conserved brain-enriched circRNA expression patterns.
- Enterprise Reuse: Establishes a reusable library preparation protocol applicable across diverse total RNA sample types.
Operational & Enterprise Impact
- Scientific Value: Improves target validation confidence through enhanced circRNA detection sensitivity and specificity.
- Operational Value: Increases reproducibility and standardization via optimized RNase R treatment and input RNA quantification.
- Strategic Value: Reduces biological risk in target selection by enabling comprehensive non-coding RNA landscape assessment.
- Portfolio Impact: Informs risk-adjusted prioritization of circRNA-associated targets based on tissue-specific expression evidence.
Implementation Considerations
- Requires expertise in RNA handling, library preparation, and circRNA bioinformatics analysis.
- Depends on access to stranded total RNA library kits, RNase R enzyme, and magnetic bead-based purification systems.
- Necessitates cross-team standardization of RNA quality metrics (RIN, DV200) and input amount titration.
- Involves adaptation considerations for low-input or degraded samples based on tissue source variability.
- Limited by circRNA detection tool variability and the need for orthogonal validation of putative junctions.
Biopharma FAQs
Why does RNase R pretreatment matter for circRNA detection?
RNase R pretreatment enriches circular RNAs by degrading linear RNA species, thereby increasing the relative abundance of circRNAs in sequencing libraries and improving detection sensitivity without introducing bias toward specific circRNA classes.
How does total RNA input amount affect circRNA library complexity?
Using four micrograms of total RNA input maximizes the diversity of detected circular RNA species, as lower input amounts reduce library complexity and limit the identification of low-abundance circRNAs across tissue types.
What quantitative measurements enable circRNA detection tool comparison?
Normalized circRNA read counts and junction-supporting read numbers from tools like find_circ, CIRI, and CIRCexplorer allow quantitative comparison of detection efficiency across library preparation conditions and tissue samples.
Why are replication requirements important for circRNA studies?
Biological and technical replicates ensure observed circRNA expression patterns are robust and not artifacts of library preparation variability, supporting reliable cross-functional interpretation in target validation pipelines.
What statistical analysis is required before implementing circRNA detection in discovery workflows?
Implementation requires differential expression analysis with appropriate multiple testing correction and reproducibility thresholds to distinguish biologically relevant circRNAs from technical noise in comparative tissue or condition studies.
