Circular RNA Sequencing (circRNA-seq)

Recent years there has been a considerable increase in interest in circRNA studies, especially circRNA expression analysis. CD Genomics provides circRNA sequencing (circRNA-seq) service for identifying circRNAs, quantifying circRNA expression levels, and characterizing their potential functions. Please contact our expert team to discuss how our circRNA-seq service can refine your NGS projects.

Overview

Circular RNAs (circRNAs) are a large class of naturally occurring endogenous non-coding RNAs (ncRNAs) that contain a covalent circular structure formed by back-splicing without a 3' tail and or 5' cap. Previous studies have shown that circRNAs are widely present in a relatively low abundance compared with linear mRNA molecules and some are conserved in eukaryotic organisms. Although the function of most circRNAs is still ambiguous, studies have revealed that circRNAs function as molecular sponges to sequester specific microRNAs (miRNAs) repress their endogenous activity. Studies also highlighted the critical roles of circRNAs in cellular metabolic processes and cancer occurrence and progression. Thus, circRNA expression analyses help interpret molecular fundamentals of complex traits and diseases. RNA sequencing (RNA-seq) has emerged as a transcriptome-wide method to characterize and quantify circRNAs. A number of computational approaches have been developed for extracting, characterizing, and quantifying circRNAs. Dissection of changes in circRNA expression among different samples is a routine analysis in circRNA studies to evaluate the significance of differentially expressed circRNAs. CD Genomics offers circRNA-seq to investigate miRNA sponges and the regulatory function in gene expression.

Features

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Mapping to genome
• Identify back-splicing junction (BSJ) reads
• circRNA identification and profiling
• Quantification and differential expression of circRNAs
• Interaction with miRNAs and functional analysis
• Target gene prediction

Sample Requirements

RNA sample (quantity ≥ 5 μg, concentration ≥ 300 ng/μL)

RIN ≥ 7.0, with smooth baseline; OD260/280: 1.8-2.2; OD260/230: ≥ 1.8.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: raw data as BAM files, coverage summary, QC report, custom bioinformatics analyses.

Demo Results

Case Studies

• • CircACTN4's Role in Intrahepatic Cholangiocarcinoma Progression

    • Background

      Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer, accounting for approximately 10-20% of all primary liver cancers and exhibiting a rapid increase in incidence over the past few decades. Understanding the pathogenic mechanisms underlying ICC is crucial for the development of effective treatment strategies. Circular RNA (circRNA) is a class of non-coding RNA known for its diverse functions, including miRNA sponging and regulation of gene expression. The role of circRNAs in ICC progression remained an area of active research.

      Methods and Key Findings

      CircACTN4 Overexpression in ICC Tissues

      • Tissue microarray analysis revealed elevated expression of CircACTN4 in ICC patient tissues, with CircACTN4 showing the highest expression among 19 upregulated circRNAs.
      • CircACTN4's resistance to RNase R digestion confirmed its circular nature.
      • High CircACTN4 expression in ICC was associated with poor 3-year overall survival rates and higher recurrence rates.

      CircACTN4 Promotes ICC Cell Proliferation and Invasion

      • Overexpression of CircACTN4 enhanced RBE cell proliferation, migration, invasion, and angiogenesis.
      • Inhibition of CircACTN4 in FRH0201 cells resulted in suppressed proliferation, migration, and invasion.
      • CircACTN4 influenced lung metastasis and tumor growth in xenograft models.

      CircACTN4 Regulates FZD7 Expression in ICC Cells

      • RNA-seq analysis identified 103 differentially expressed genes in RBE cells overexpressing CircACTN4, with Hippo/Wnt-related genes highly enriched.
      • CircACTN4 affected the protein levels of FZD7, ID2, CCN2, and AXIN2.
      • TCGA data showed significantly higher FZD7 expression in ICC tissues.
      • This result suggests that CircACTN4 may impact FZD7 expression.

      Interaction Between CircACTN4 and YBX1

      • RNA-pull down and western blotting experiments demonstrated the interaction between CircACTN4 and YBX1.
      • TCGA data showed elevated YBX1 expression in ICC tissues, suggesting its role in ICC progression.

      CircACTN4 Recruits YBX1 to Activate FZD7 Transcription

      • ChIP-seq analysis showed widespread YBX1 binding sites in FRH0201 cells, with 24.88% located in promoter regions.
      • YBX1 binding motif analysis revealed different sequence preferences.
      • CircACTN4 and YBX1 together activated the transcription of the FZD7 gene.

      CircACTN4 Upregulates YAP1 Expression by Sponging miR-424-5p

      • CircACTN4 acted as a sponge for multiple miRNAs, but only miR-424-5p was upregulated when CircACTN4 was silenced.
      • RNA-RIP experiments confirmed the direct binding of CircACTN4 to miR-424-5p.
      • The inhibition of miR-424-5p reduced cell proliferation, migration, and invasion, along with decreased YAP1 levels.

      CircACTN4 Involvement in Wnt/Hippo Signaling

      • CircACTN4 influenced the expression of β-catenin and p-GSK3b in ICC cells.
      • CircACTN4 promoted YAP1 and β-catenin interaction and their nuclear accumulation.
      • Wnt/β-catenin pathway activity correlated with CircACTN4 expression.

FAQ

• • What is the minimum amount of Total RNA required for submission?

    • We recommend a minimum of 5 µg of Total RNA for each submission. For detailed submission guidelines, please refer to our Sample Submission Recommendations.

• • How much circRNA sequencing data is recommended?

    • It is generally recommended to generate at least 10 GB of sequencing data to ensure sufficient coverage for circRNA analysis, based on saturation analysis.

• • Is biological replication necessary for circRNA sequencing?

    • Yes, it is essential. We recommend a minimum of three biological replicates, although more replicates are beneficial for robust results.

• • What's the difference between lncRNA data analysis and circRNA standard library preparation (Rnase enrichment) analysis?

    • In lncRNA data (10 GB clean data), typically, you can identify around 1,000 to 2,000 circRNAs. In contrast, the Rnase enrichment method can identify 20,000 to 30,000 circRNAs, with data quantities in the tens of thousands. We recommend these approaches based on your research goals. If your study is exploratory, lncRNA library sequencing offers cost-effectiveness. For targeted circRNA research, the standard circRNA library preparation method is recommended.

• • What are the methods for circRNA validation?

    • CircRNA can be validated through two methods:

      • Quantitative Validation: Design primers based on junction sites and perform qPCR validation.
      • Functional Validation: Employ methods like circRIP for miRNA sponge functionality validation, or simulate functional validation through miRNA knockdown and antagonists.
• • Are circRNAs completely stable and resistant to degradation?

    • CircRNAs are known for their resistance to RNase R degradation. However, degradation can occur through two processes: RNase R degradation and hydrolysis. Hydrolysis affects both circular and linear RNA and can occur more readily in circular RNA due to the close proximity of base pairs. To maintain circRNA stability, they should be stored at low temperatures and away from magnesium ions. While RNase R degradation can enhance sensitivity in circRNA detection, it may not improve specificity.

• • Is it preferable to use microarray or sequencing for circRNA analysis?

    • We recommend using sequencing technology for circRNA analysis as it enables the discovery and identification of new results.

• • Are circRNA differences significant between samples?

    • Yes, circRNA levels can vary significantly between samples. Unlike most genes, circRNAs are not part of the "housekeeping" genes and should not be normalized in the same manner. Their expression levels can exhibit substantial differences between samples.

• • Can circRNA sequencing be combined with other sequencing technologies?

    • Yes, circRNA research can benefit from combining with other sequencing techniques. For example, you can study the interaction of specific RNA-binding proteins (RBPs) with circRNAs using RIP-seq. At the genome level, ATAC-seq and ChIP-seq can help explore the transcriptional activity and regulatory elements influencing circRNAs. These approaches can also assess whether linear host RNA and circRNA share similar transcriptional regulation.

References:

1. Zhang J, Chen S, Yang J, et al. Accurate quantification of circular RNAs identifies extensive circular isoform switching events. Nature communications, 2020, 11(1): 1-14.
2. Xiong D, Feng Z, Lai Z, et al. High throughput circRNA sequencing analysis reveals novel insights into the mechanism of nitidine chloride against hepatocellular carcinoma. Cell death & disease, 2019, 10(9): 1-16.
3. Liu Z, Ran Y, Tao C, et al. Detection of circular RNA expression and related quantitative trait loci in the human dorsolateral prefrontal cortex. Genome biology, 2019, 20(1): 1-16.
4. Chen, Qinjunjie, et al. "Circular RNA ACTN4 promotes intrahepatic cholangiocarcinoma progression by recruiting YBX1 to initiate FZD7 transcription." Journal of hepatology 76.1 (2022): 135-147.