Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Real-time PCR assay for detecting illicit steroid administration in veal calves allows reliable biomarker profiling of formalin-fixed, paraffin-embedded (FFPE) archival tissue samples Alessandro Benedetto a , Marzia Pezzolato a, ⁎ , Chiara Beltramo a , Valentina Audino a , Francesco Ingravalle a , Caterina Pillitteri b , Stefano Foschini c , Simone Peletto a , Elena Bozzetta a a Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154 Torino, Italy b Direzione Generale Sanità Regione Piemonte - Settore Prevenzione e Veterinaria, Corso Regina Margherita 153 bis, 10152 Torino, Italy c Direzione Generale Sanità Regione Lombardia, Piazza Città di Lombardia n. 1, 20124 Milano, Italy ARTICLE INFO Keywords: Sex steroids Illicit treatment FFPE Transcriptional Biomarker Standardization RNA Veal calves ABSTRACT In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and opti- mization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit admin- istration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee. 1. Introduction In response to the European Food Safety Authority (EFSA) call for a comprehensive estimation of growth promoter abuse in cattle meat production (EFSA, 2013), various screening methods have been devel- oped to reveal such illicit practices, which are often based on the combined use at low dosages of a broad range of substances (e.g., sex steroids, glucocorticoids, beta-agonists). Several novel screening methods work by detecting modulations of selected biomarkers (Bozzetta et al., 2011; Guglielmetti et al., 2014; Ludwig, Van Ginkel, & Nielen, 2014; Pezzolato et al., 2013; Richelmi et al., 2017; Riedmaier, Spornraft, & Pfaffl, 2014). Since 2008 a histological method to reveal microscopic lesions related to illicit steroid treatment has been applied in the frame of Italian National Residue Control Plans (NCRPs). Among other biomarker based methods, genome-wide analysis of the transcriptome has helped researchers identify multiple differen- tially-expressed gene (DEG) patterns (signature patterns) related to growth promoter abuse (Pegolo et al., 2015; Riedmaier et al., 2012). Moreover, the discovery of promising biomarkers has been advanced by proper statistical data treatment to limit confounding variance in- troduced by species variability, inter-individual variability, experi- mental design and/or sampling procedures (Riedmaier et al., 2014). However, limitations in the standardization, validation, and cost ana- lysis of omic-techniques (e.g., to investigate whole transcriptome, pro- teome, lipidome, metabolome) have hindered their applicability as everyday diagnostic tools, prefiguring them to the more suitable roles of biomarker discovery. Only recently some applications of untargeted omic-techniques become available as potential confirmatory analysis for the control of growth promote abuse (Dervilly-Pinel et al., 2018). Molecular tests based on the analysis of a few specific biomarkers can often be limited to the detection of single illicit substance and limited to a single organ and/or few target tissues, yet they are still considered more appropriate for cost-effective screening of large sample sizes (Cucuzza et al., 2017; Divari et al., 2013; Starvaggi Cucuzza, Biolatti, Scaglione, & Cannizzo, 2018). Nevertheless, in a “real life” monitoring strategy, especially when working with RNA biomarkers (mRNA, miRNA, etc.), the use of such molecular methods falls short of sample collection and storage https://doi.org/10.1016/j.foodchem.2019.126061 Received 5 February 2019; Received in revised form 14 November 2019; Accepted 13 December 2019 ⁎ Corresponding author at: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154 Torino, Italy. E-mail address: marzia.pezzolato@izsto.it (M. Pezzolato). Food Chemistry 312 (2020) 126061 Available online 19 December 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved. T