Workflow for the identification and validation of microRNAs: the colorectal cancer experience

Abstract

MicroRNAs are promising non invasive diagnostic biomarkers that can be easily detected in plasma. However, several factors, both pre-analytical and analytical, must be taken into account during the identification and validation of miRNA biomarkers.The workflow for the identification of a biomarker should start with a discovery phase, ideally based on high-throughput technologies to identify candidate biomarkers that will be further investigated in the subsequent validation phases[1].The discovery phase should also allow the setting-up of all the aspects of the pre-analytical phase of sample collection/processing and the pre-processing steps of the data, such as data normalization[2].During validation phase(s) the performance of the candidate biomarkers should be adequately explored using advanced statistical methodologies to identify powerful miRNA-based signatures[1]. Assay-oriented step(s) may be included in the workflow to set-up new assays easy transferable to the clinical setting. In this workflow the availability of stored material could be of help in the biomarker development pipeline. As part of the ongoing EDERA project (http://www.ederaproject.it) at Istituto Nazionale Tumori (INT), we applied the described workflow for the identification and validation of miRNAs for the early detection of colorectal cancer, using individuals positive to the fecal occult blood test as target population. The discovery phase was performed on retrospective plasma samples stored in our biobank; miRNAs profiling was done using the TaqMan\uae Array Human MicroRNA Card A (Applied Biosystems). The validation phase consisted of two phases based on plasma samples prospectively collected at INT and during a multi-centric study. [1]Verderio P,et al.BJC.2016.doi:10.1038/bjc.2016.164[in press] [2]Verderio P,et al.Anal Biochem.2014;461:7-