Contents lists available at ScienceDirect International Journal of Biochemistry and Cell Biology journal homepage: www.elsevier.com/locate/biocel Lung inflammation after bleomycin treatment in mice: Selection of an accurate normalization strategy for gene expression analysis in an ex-vivo and in-vitro model Veronica Della Latta a,1 , Manuela Cabiati a,1 , Silvia Burchielli b , Giada Frenzilli c , Margherita Bernardeschi c , Antonella Cecchettini c , Federica Viglione a , Maria-Aurora Morales a , Silvia Del Ry a, ⁎ a CNR Institute of Clinical Physiology, Laboratory of Biochemistry and Molecular Biology, Pisa, Italy b Fondazione Gabriele Monasterio, Pisa, Italy c University of Pisa, Dept. Experimental and Clinical Medicine, Pisa, Italy ARTICLE INFO Keywords: Pulmonary fibrosis Bleomycin Reference genes Real-Time PCR PTX-3 TNF-α ABSTRACT Pulmonary fibrosis (PF) is the most common and aggressive interstitial lung disease, characterized by a patchy development of fibrosis leading to progressive destruction of the normal lung architecture which is preceded by an inflammatory process. Gene expression studies are important to understand the development of PF but the accuracy and reproducibility of Real-Time PCR depend on appropriate normalization strategies. This study aimed to analyze the expression variability of eight commonly used reference genes during the initial inflammatory phase of bleomycin-induced PF in a mouse model and to verify whether the selected reference genes could be applied to an in-vitro model of BLM-treated primary murine lung fibroblasts. Wild-type C57BL/6 mice (n = 40) were used. Real-Time PCR was carried out on lung tissue of mice either BLM (BLM-tm) or physiological solution-treated (PSS-tm), and in primary lung fibroblasts, isolated from healthy C57BL/6 mice. Histological analysis was performed to confirm the inflammation development. During inflammation, the most stable genes resulted: PPIA, HPRT-1 and SDHA for both models; the normalization strategy was tested analyzing mRNA expression of PTX-3 and TNF-α which resulted up-regulated both in ex-vivo and in-vitro with respect to PSS-tm/fibroblasts. Histological analysis supported the results. This study identified a new set of reference genes expressed both in the in-vitro and ex-vivo models. A higher expression of both markers in BLM-tm with respect to PSS-tm indicated that BLM might lead to increased PTX-3 local production by a co-regulation with TNF-α at lung level. 1. Introduction Interstitial lung diseases (ILDs) are a heterogeneous non-neoplastic group of more than 200 different diseases with variable etiology, such as autoimmunity, medications, radiation or exposure to substances (e.g. asbestos, coal, silica). They are characterized by different degrees of fibrosis and inflammation leading to progressive destruction of the normal lung architecture (Demedts et al., 2001), since the interstitium, the airspaces, peripheral airways, and vessels along with their respec- tive epithelial and endothelial linings are affected (American Thoracic Society, 2001). In this context, idiopathic pulmonary fibrosis is the most aggressive interstitial lung disease associated with the histological appearance of usual interstitial pneumonia on lung biopsy. Pulmonary fibrosis (PF) is characterized by cellular proliferation and progressive accumulation of extracellular matrix constituents resulting in remodel- ing of the lung interstitium (Selman et al., 2001; Thannickal et al., 2004; Gross and Hunninghake, 2001; Raghu et al., 2011). To date, the molecular mechanisms and potential genetic pathways responsible for PF development have not been yet identified, although inflammation seems to be one of the leading causes of disease initiation and progression (Selman and Pardo, 2002; Scotton and Chambers, 2007; Della Latta et al., 2015). The murine model represents the most widely used animal model to study the fibrotic process. (Moeller et al., 2006; Moore and Hogaboam, 2008; Degryse and Lawson, 2011). Different approaches have been used to induce PF. Bleomycin (BLM), an anti-neoplastic drug, is commonly http://dx.doi.org/10.1016/j.biocel.2017.05.016 Received 14 September 2016; Received in revised form 10 April 2017; Accepted 8 May 2017 ⁎ Corresponding author at: CNR Institute of Clinical Physiology, Via Giuseppe Moruzzi 1, 56124, Pisa, Italy. 1 Contributed equally to this work. E-mail address: delry@ifc.cnr.it (S. Del Ry). International Journal of Biochemistry and Cell Biology 88 (2017) 145–154 Available online 16 May 2017 1357-2725/ © 2017 Elsevier Ltd. All rights reserved. MARK