The mRNA was extracted from explants using TRI Reagent® (T9424; Sigma-Aldrich, St Louis, MO, USA.) according to the manufacturer’s instructions. The mRNA quantification was assessed using a Nanodrop system (ND 200C; Fisher Scientific, Hamton, PA, USA), and mRNA quality was evaluated by visualization of 28S and 18S rRNA bands after electrophoresis of a 1.5% red staining agarose gel (41,003; Biotium, Hayward, CA, USA). The synthesis of cDNA was performed using M-MLV reverse transcriptase enzyme (M5313; Promega, Madison, WI, USA) from 1 μg of total RNA in a 20 μL reaction volume using oligo (dT) primer (C1101; Promega, Madison, WI, USA).
The validation of reference ribosomal protein L32 (RPL32) and target COL1A2 genes was performed as described by Amaral et al. [15 (link)]. The equine-specific primer sequences are listed inTable 1 . The qPCR reactions of both genes were run in duplicate in the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Warrington, UK) in a 96-well plate (4306737; Applied Biosystems, Warrington, UK) and product specificity was analyzed, as previously described [15 (link),52 (link)].
The validation of reference ribosomal protein L32 (RPL32) and target COL1A2 genes was performed as described by Amaral et al. [15 (link)]. The equine-specific primer sequences are listed in
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