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Singh et al. Cancer Drug Resist. 2025;8:56 Page 11 of 20

Figure 3. Schematic representation of the experimental and analytical pipeline for circRNA detection and characterization. (1) Sample
collection: blood is collected in RNase-free EDTA tubes, followed by rapid centrifugation to obtain plasma; (2) Exosome and RNA isolation:
performed using ultracentrifugation or commercial high-sensitivity RNA extraction kits; (3) CircRNA enrichment: strategies to enrich
circRNAs for downstream analyses; (4) Detection methods: include qRT-PCR (sensitive, rapid), ddPCR (absolute quantification), and
RNA-seq (global profiling); (5) Bioinformatic tools: circRNA analysis pipelines such as CIRCexplorer2, find_circ, and CIRI2, along with
databases such as circBase, circRNADb, and CircInteractome; (6) Data analysis and interpretation: application of circRNA data in clinical
translation and biomarker development [Created in BioRender. Singh DD (2025)]. circRNA: Circular RNA; EDTA:
ethylenediaminetetraacetic acid; qRT-PCR: quantitative reverse transcription polymerase chain reaction; ddPCR: droplet digital
polymerase chain reaction; RNA-seq: RNA sequencing.

enabling real-time therapy adjustments based on evolving resistance patterns identified non-invasively .
[6]
Achieving this will require multi-center collaboration, standardized protocols for circRNA extraction and
quantification, and regulatory support for biomarker-based decision making. As precision oncology
advances, circRNA liquid biopsies represent a promising tool for early resistance detection and optimized
therapy across multiple cancer types.

LIMITATIONS OF THE STUDY ON CLINICAL TRIALS ON LIQUID BIOPSY-BASED DETECTION OF

CIRCULATING CIRCRNAS FOR TRACKING DRUG RESISTANCE IN CANCER
Despite the growing interest in utilizing liquid biopsy for cancer management, clinical trials specifically
focused on the detection of circulating circRNAs for monitoring drug resistance remain limited and face
several important challenges [114] . One of the foremost limitations is the lack of ongoing or completed
large-scale clinical trials that investigate the diagnostic or prognostic utility of circRNAs in a real-world
therapeutic context [115] . Most available studies are preclinical or translational, primarily using in vitro cell
lines or small patient cohorts, which limits their generalizability and clinical applicability [116] . The lack of
robust clinical validation and longitudinal patient data makes it difficult to establish circRNAs as reliable
biomarkers for tracking drug resistance. Additionally, the standardization of circRNA detection protocols
presents a major barrier . There is currently no universally accepted method for the enrichment, isolation,
[117]
and quantification of circRNAs in clinical samples [118] . Techniques such as RNase R treatment, divergent
primer design, and RNA seq are highly sensitive to experimental conditions, leading to variability in results
across laboratories and studies [119] . Furthermore, the low abundance and tissue-specific expression of
circRNAs often require advanced technologies such as ddPCR or deep RNA seq, which may not be available

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