A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

Background Recently, many potential non-invasive biomarkers have been developed using exosome-based liquid biopsy for early detection, diagnosis, and treatment of cancer. However, exosome analyses from small liquid biopsy samples have been limited with regard to sensitivity and efficiency. Methods We used a breast cancer model mouse with ERBB2 overexpression and collected liquid biopsy samples. We performed two-stage PCR for ERBB2, PPP1R1B, GRB7, and STARD3 genes. We compared conventional PCR methods with our two-stage PCR method. Both methods had same step of primer and cycle and amplification condition for RT-qPCR. PCR amplicons quality was validated by using the Sanger method. Results Quantitative analysis of all samples by conventional PCR compared with two-stage PCR template dilution (1/200) showed similar Ct values in all sample groups (cell, supernatant, urine, and plasma). Sequencing of the two-stage PCR-derived products revealed no changes in product sequence compared to that of conventional PCR. The obtained sequences showed 98-99% match of target gene sequences in the databases according to BLAST searches. Conclusion Two-stage PCR has better sensitivity and efficiency than conventional PCR methods for small-volume samples. Additionally, this method is useful for monitoring, as many genes can be assayed at once.