qPCR Assay for T-DNA (HptII) and Repair Template (mCherry) Copy Number Determination

Hydrolysis probe-based quantitative real-time PCR (qPCR) was used to determine copy number of the T-DNA (HptII) and repair template (mCherry) in transgenic barley lines. The reaction compared the Cq values of an HptII amplicon to a single-copy barley gene CO2 (Constans-like, {"type":"entrez-nucleotide","attrs":{"text":"AF490469","term_id":"21667474","term_text":"AF490469"}}AF490469) amplicon and the Cq values of an mCherry amplicon to a single-copy barley gene CO2 (Constans-like, {"type":"entrez-nucleotide","attrs":{"text":"AF490469","term_id":"21667474","term_text":"AF490469"}}AF490469) amplicon within FAM/VIC duplexed assays (see Supplementary Table 6). The reactions used Thermo ABGene Absolute qPCR Rox Mix (Cat. number AB1139) with the probes and primers at final concentrations of 200 nM (HptII and mCherry) and 100 nM (CO2). The assay contained 5 μl DNA solution and was optimised for DNA concentrations of 1–10 ng/μl (5–50 ng DNA in the assay). PCRs were carried out as 25 μl reactions in a Bio-Rad CFX96 machine (C1000 Touch, Bio-Rad, Hercules, CA, USA). The detectors used were FAM-TAMRA and VIC-TAMRA. The PCR cycling conditions were 95°C for 15 min (enzyme activation), 40 cycles of 95°C for 15 s, and 60°C for 60 s. Cq values were determined using CFX96 software (version 3.1), with Cq determination set to regression mode. Values obtained were used to calculate copy number according to published methods (Weng et al., 2004).