A. Sidabraite1, U. Ahmed2, S.K. Jones2, A.K. Gulla3 1Vilnius University, Faculty Of Medicine, Vilnius, VILNIUS, Lithuania 2Vilnius University, VU LSC-EMBL Partnership For Genome Editing Technologies, Life Sciences Center, Vilnius, VILNIUS, Lithuania 3Vilnius University, Faculty Of Medicine, Institute Of Clinical Medicine, Vilnius, VILNIUS, Lithuania
Introduction: Cholangiocarcinoma (CCA) includes heterogeneous tumours with complex diagnoses, management, and poor prognoses. Diagnosing distal CCA (dCCA) is particularly challenging in clinical cases where it presents a diagnostic dilemma due to its clinical and imaging similarities to pancreatic ductal adenocarcinoma (PDAC) and benign diseases. Current diagnostic methods are inadequate, necessitating the development of more sensitive, specific, and non-invasive biomarker detection techniques. MicroRNAs show promise as biomarkers, and innovative approaches like CRISPR/Cas9-mediated detection may enhance diagnostic capabilities.
Methods: We propose using Rolling Circle Amplification (RCA)-assisted CRISPR/Cas9 cleavage (RACE) to detect miRNA-16 and miRNA-877 panel in exosomes derived from plasma samples. Exosomes will be isolated from patients with CCA, PDAC, benign diseases, and healthy controls. The miRNAs will be analyzed using RACE and results will be compared to those obtained via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RACE method involves two key steps: replication, where a long double-stranded DNA (dsDNA) assembly is generated through the interaction between the padlock probe with the PAM domain and the target miRNA; and detection, where the sgRNA/Cas9 complex cleaves the dsDNA, leading to a measurable fluorescence signal.
Results: We anticipate that RACE will exhibit superior sensitivity and specificity compared to RT-qPCR, or at least show high consistency, while offering advantages in processing time and cost-effectiveness.
Conclusion: RACE technology has the potential to significantly improve miRNA detection, offering clinicians a robust tool for differentiating dCCA from other diseases with similar clinical and radiological presentations. This method could reduce the reliance on invasive biopsies, ultimately enhancing patient outcomes through earlier and more accurate diagnosis.