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Introduction: Covalently closed, single-stranded circular RNAs (circRNAs) are a distinct class of RNA that lack free 5′ caps and 3′ poly(A) tails and are resistant to nucleases, making them more stable than conventional linear RNAs. With the rapid advancement of high-throughput sequencing and bioinformatics technologies, tens of thousands of circRNAs have been identified across diverse species. Most of these circRNAs exhibit conservation and stability among different organisms, and their expression is characterized by cell‑, tissue‑, and developmental stage‑specificity. In addition to the well‑known ceRNA regulatory mechanism—where circRNAs competitively bind microRNAs—circRNAs also harbor novel functions, such as translating peptides, and hold tremendous potential for both basic research and translational applications in medicine, as well as in plants and animals.
Initial Sample Amount and Submission Recommendations
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Sample Type |
Initial amount |
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Animal and clinical organ tissues, such as brain tissue, etc. |
>20mg |
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Animals and clinical skin/bone/vascular/fat tissues, etc. |
>100mg |
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Plant leaf tissue/flower |
>200mg |
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Plant roots/stems/fruits/seeds |
>500mg |
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Primary cells/cell lines |
>5×10^6 units |
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Neutrophils/Eosinophils/Basophils |
>5×10^7 units |
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Exosome sample |
>1×10^8 units |
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Serum/plasma/cerebrospinal fluid/joint effusion/follicular fluid |
>2mL |
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Cell culture supernatant |
>20mL |
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Urine |
>30mL |
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Total RNA |
>1 μg and RIN > 7.0 |
Precautions:
① Tissue samples are recommended to be stored in RNAlater, RNAHold, RNAProtect, or other suitable tissue preservation solutions, and then either frozen at −80°C or shipped on dry ice.
② After thoroughly lysing the cell samples with a lysis buffer such as TRIzol, store them at −80°C or ship them on dry ice.
③ For more detailed sample preparation guidelines, please contact online customer service.
Bioinformatics Analysis Workflow and Analytical Content
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circRNA |
Analysis Content |
Remarks |
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Sequencing data quality control |
Raw Data Quality Control Statistics Table |
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Reference genome alignment analysis |
Reference Genome Gene Statistics Table |
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Reference Genome Alignment Reads Statistics Table |
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Table of Statistics on the Number of Identified circRNAs in the Sample |
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Statistical Chart of the Distribution of Reference Genome Alignment Regions |
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Statistical chart of the distribution of nonlinear alignment regions in the reference genome |
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circRNA identification and quantitative analysis |
circRNA fitted sequence |
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circRNA expression profile |
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BSJ Statistics Table for circRNAs |
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circRNA statistical analysis |
Pie chart of circRNA type statistics |
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Box plot of circRNA expression distribution statistics |
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Density plot of circRNA expression levels |
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Statistical chart of circRNA chromosomal distribution |
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Statistical chart of the number of exons in circRNAs |
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Chart of the number of parental gene-derived circRNAs |
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circRNA differential analysis |
Histogram of the number of differential circRNAs |
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Differential expression analysis of circRNAs in the Case vs. Control comparison group |
CircRNA Volcano Plot for the Case vs. Control Comparison Group |
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CircRNA heatmap of the Case vs. Control comparison group |
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Case vs. Control comparison group circRNA scatter plot |
Application Scenarios and Case Studies
Application Scenario 1: Structural molecule–mediated recruitment of core proteins and nucleic acids
Scope of application: Any research area, including basic medicine, biochemistry, and molecular biology.
LncRNAs and circRNAs can, at times, function as specialized RNA molecules that serve as scaffolds for protein–protein complexes or protein–nucleic acid assemblies, thereby exerting a range of regulatory effects on downstream molecules. For example, certain circRNAs recruit ubiquitin‑E3 ligases to mediate the degradation of target proteins, while some lncRNAs recruit transcription factors to bind to the promoter regions of specific genes, thereby activating their expression.
Application Scenario 2: Exosomes
Scope of application: Any research area, including clinical and translational medicine, basic medicine, and others.
Because lncRNAs in exosomes exist as fragmented pieces, these random fragments not only hinder detection but also, combined with their low abundance and poor reproducibility, have kept lncRNAs from becoming a mainstream focus in exosome research. In contrast, circRNAs, owing to their remarkable stability, can be isolated from exosomes in biofluids and serve as biomarkers for disease; moreover, they hold promise for downstream applications such as exosome‑based therapeutics, making them one of the most actively pursued subfields in the post‑ceRNA era. One major avenue involves identifying disease‑specific biomarkers, typically supported by large‑scale cohort studies. Another focuses on translational and mechanistic investigations, encompassing the molecular mechanisms underlying exosome–target cell interactions as well as therapeutic strategies.
Application Scenario 3: Peptide Translation
Scope of application: Any research area, including clinical and translational medicine, basic medicine, biochemistry, and molecular biology.
A segment of untranslated RNA on circRNAs, known as an IRES, can fold into a structure resembling the initiator tRNA and recruit additional ribosomes. Under normal conditions, the IRES does not bind to eIFs but instead associates with a class of proteins called ITAFs. These ITAFs facilitate the recruitment of ribosomes to the internal structure of the circRNA, thereby initiating protein translation. Furthermore, IRES enhancers may also be present, similar to UTR elements found in circ‑ZNF609. Additionally, studies employing ribosome profiling and transcriptome sequencing have revealed that the noncoding RNA LINC‑PINT, upon forming a circular RNA, can be translated into functional peptides.
Application Scenario 4: ceRNA Regulatory Mechanism
ceRNA stands for competing endogenous RNA, a type of RNA that can competitively bind to RNA‑binding elements. Typically, lncRNAs and circRNAs compete to bind miRNAs; accordingly, we often refer to lncRNAs and circRNAs as ceRNAs. The ceRNA regulation network, or ceRNAregulation network, denotes the entire regulatory cascade involving ceRNAs. CeRNA analysis refers to the systematic examination of such a ceRNA regulatory network, commonly encompassing either circRNA–miRNA–mRNA or lncRNA–miRNA–mRNA interactions.
Project Workflow
HeYuan Bio’s eukaryotic reference‑based transcriptome sequencing offers researchers a comprehensive end‑to‑end service pipeline—covering sample extraction, library construction and sequencing, and data analysis—delivering high‑quality results and providing robust evidence to support downstream research.
Original article link: HeYuan Bio – circRNA Sequencing
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