Optimized Workflow for Sensitive Detection of Circulating Tumor Cells Using qPCR and Pre-Amplification to Minimize Cross-Contamination.

Circulating tumor cells (CTCs) offer valuable insights for cancer treatment, but their small number, low abundance and heterogeneity pose challenges for routine clinical use. The detection of CTCs requires sensitive technologies to overcome the abundance of hematogenous cells. This typically involves enriching the sample to increase the relative amount of CTCs, followed by analytical methods like gene expression profiling. Quantitative PCR (qPCR) has proven useful for assessing CTC heterogeneity and molecular characterization. However, ensuring accurate results requires high CTC abundance and minimal contamination from leukocytes. We developed a workflow using microfluidic CTC enrichment and qPCR to detect specific gene transcripts, utilizing a pre-amplification protocol to enhance the signal. To minimize cross-contamination risks, we employ deoxyuridine triphosphate (dUTP) in the pre-amplification step and a heat-labile uracil-N-glycosylase (UNG) enzyme, which prevents false positives. This approach ensures sensitive, accurate CTC detection while reducing contamination risk, making it a robust method for clinical application.