Moving from Discovery to Validation in Circulating microRNA Research

Abstract

This report highlights the need to add a technical validation step to the high-throughput-based miRNA identification workflow, after their discovery and before the validation step in an independent series.

Plasma miRNA‐based signatures in CRC screening programs

“…In the discovery phase, 60 plasma samples from FIT+ subjects (50% male, median age at blood collection 61 years, range 51-69 years) were profiled. The percentages of each lesion were 21.67% for LgA (median age at blood collection 60 years, range 51-68 years), 25.00% for HgA (median age at blood collection 62 years, range 51-68 years), 16.67% for CL (median age at blood collection 63 years, range 51-68 years) and 36.67% for NL (median age at blood collection 60 years, range 51-69 years) ( Table 1). Using the updated version of the normalization quantitative polymerase chain reaction array algorithm, we identified a subset of four miRNAs (hsa-miR-30c-5p, hsa-miR-345-5p, hsa-miR-150-5p and hsa-miR-296-5p) that best resembled the results with the overall mean approach, reflecting "substantial agreement" according to Landis and Koch classification criteria.…”

“…The same custom card format was used for IVC, while EVC was conducted using Custom TaqMan Array microRNA cards designed in 16 format (15 miRNA assays and the small nuclear RNA U6 predefined control assay). Quantitative real‐time polymerase chain reaction and data acquisition were as in Verderio et al …”

“…In the discovery phase, the miRNA profile of each sample was analyzed using the TaqMan Array Human MicroRNA A Cards v2.0 (ThermoFisher Scientific) containing 377 human miRNAs as described in Zanutto et al ., and Verderio et al . For the technical validation phase, Custom TaqMan Array microRNA cards in 24 format (23 unique miRNA assays and a predefined control assay, the small nuclear RNA U6) were designed as described by Verderio et al . The same custom card format was used for IVC, while EVC was conducted using Custom TaqMan Array microRNA cards designed in 16 format (15 miRNA assays and the small nuclear RNA U6 predefined control assay).…”

Developing miRNA signatures: a multivariate prospective

“…The process begins with the discovery, and is followed by a validation phase and, finally, the clinical application of the identified biomarker signature ( Verderio et al , 2010 ). Two additional assay set-up steps could be included in the workflow, before (assay optimisation) and/or after (assay development) the validation phase ( Verderio et al , 2015 ). Independent cohorts of patients from the same target population should be considered for the discovery and validation phases.…”

“…In conclusion, although it was acknowledged that availability of standardised operative procedures (SOPs) for both the pre-analytical and analytical phase is fundamental for miRNA reliability ( Verderio et al , 2010 , 2015 ), SOPs are not sufficient to address clinical utility. An additional requirement for developing multivariate prediction models based on miRNAs is the optimisation of the statistical analysis workflow, involving the complete procedure, from the generation of the initial multivariate model to the final one.…”