Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary...

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAX^TM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.