Sybyr gold

Total RNA of FACS-purified HCT116 transfectants was prepared using RNeasy Mini kits (Qiagen). RT-qPCR analysis was performed using PrimeScript High Fidelity RT-PCR kit (Takara Bio Inc) with SYBYR Gold (Thermo Fisher Scientific) and a CFX96 Real-Time system instrument (Bio-Rad, Hercules, CA, USA).

Total RNA was immediately prepared from leaves and petals of wild type (WT) plants, and eYGFPuv- and eYGFP-expressing plants using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. First-strand complementary DNAs (cDNAs) were synthesized by the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan), in accordance with the manufacturer’s instructions. For real-time PCR, we used KOD Dash DNA polymerase (Toyobo) supplemented with SYBYR Gold (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed on a CFX96 Real-Time system instrument (Bio-Rad), using the primer pairs listed in Supplementary Table S5. Because most reagents for measuring the activity of anti-oxidative enzymes exhibit a certain fluorescence overlapping that of transgenes^{40 (link),41 (link)}, we only measured the mRNA levels of genes coding for the typical anti-oxidative enzymes superoxide dismutase (SOD) and catalase (CAT)^{30 (link)}. Transcript levels of fluorescence genes and anti-oxidative enzyme genes were normalized according to that of cyclophilin (CYP), as this gene is more suitable than the conventional GAPDH due to its stable expression regardless tissue or cell specificity, morphogenetic process, and environmental conditions^{28 (link)}.