Profiling circulating miRNA from liquid biopsies
tumor, tumour
TUMOR, TUMOUR
The miRNAs will be extracted from 200 μl of cell-free plasma using the miRNeasy serum/plasma extraction kit (Qiagen) according to the manufacturer’s instructions. RNA quality will be assessed using a Nanodrop Spectrophotometer (Thermo Scientific), resulting in a mean 260/280 ratio of 1.95. RNA will be processed for miRNA-sequencing and RT-qPCR. The sequencing of miRNAs extracted from the serum through liquid biopsy will be performed by Qiagen, a leading provider for RNA studies. The study will include sample quality control using qPCR, library construction and quality control, miRNA sequencing, and analysis of the results.As a complement, digital PCR (dPCR) based on the partitioning of the sample into thousands of micro-reactions of defined volume will be performed with miRNA of interest because of higher precision and sensitivity to detect low-abundance miRNAs in plasma samples [The potential predictive value of miRNAs signature will be validated. In addition, optimal expression cutoff values will be determined using receiver operating characteristic (ROC) curve analysis to determine the relationship between miRNAs signature and Overall Response Rate (ORR; used as an endpoint). Hazard ratios will be adjusted in multivariate models including the principal known confounding prognostic factors, such as patient age, tobacco smoking, treatment, co-morbidities, margin status, extra-capsular status, tumour localization, differentiation, stage, lymph node involvement, and tumor size.