Quantifying IRC7 Gene Copy Numbers

The relative copy numbers of the IRC7 gene in selected strains were estimated with quantitative PCR of genomic DNA. Primers PF6 and PR7 from Roncoroni et al. (2011 (link)) were used for IRC7. Copy numbers were normalized to that of ALG9 and UBC6 (primers listed in Krogerus et al. (2019 (link))). The efficiencies (E) of the qPCR assays (ranging from 1.9 to 1.94) for each primer pair were calculated using the formula 10(− 1/m), where m is the slope of the line of the threshold cycle (CT)-versus-log dilution plot of the DNA template (8 pg to 8 ng input DNA) (Pfaffl 2001 (link)). The qPCR reactions were prepared with PerfeCTa SYBR® Green SuperMix (QuantaBio, Beverly, MA, USA) and 0.3 μM of the primers. The qPCR reactions were performed on a LightCycler® 480 II instrument (Roche Diagnostics, Basel, Switzerland) in four technical replicates on 1 ng template DNA. The following programme was used: pre-incubation (95 °C for 3 min), amplification cycle repeated 45 times (95 °C for 15 s, 60 °C for 30 s, 72 °C for 20 s with a single fluorescence measurement), melting curve programme (65–97 °C with continuous fluorescence measurement), and finally a cooling step to 40 °C. The copy numbers of IRC7 relative to ALG9 and UBC6 were calculated using the Pfaffl method (Pfaffl 2001 (link)).