For qRT-PCR analyses of homozygous lines, total RNA was isolated from three biological replicates using the NucleoSpin RNA kit (Machery-Nagel, Düren, Germany). DNA was removed by an on-column treatment with rDNase (Machery-Nagel, Düren, Germany). 2–3 μg total RNA were reverse transcribed with M-MuLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) using an oligo(dT) primer. The cDNA equivalent of 30–50 ng total RNA was used in a 12 μl PCR reaction with SsoAdvancedUniversal SYBR Green Supermix (BioRad, München, Germany) in a CFX96 Real-Time System Cycler (BioRad, München, Germany). The relative quantification was calculated with the ΔΔCt method using ACT8 (AT1G49240) as a standard (Pfaffl, 2001). For the analysis of the pooled independent T2 transgenic lines, a quarter of all available independent lines per construct (18 to 45) was sampled resulting in four replicate pools comprising between four and eleven lines. Around 1000 seeds were used for pooling per line. One RNA sample was extracted per replicate pool and processed as described above.