Gene Expression Analysis by RT-qPCR

Arabidopsis plants were transferred to LL conditions for 26 h, then harvested every 4 h for 48 h. Harvested tissues were instantly frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using TRIzol Reagent (Invitrogen) followed by RNase-free DNase treatment (Qiagen). cDNA was synthesized from 2 µg of total RNA using random hexamer primers and SuperScript II Reverse Transcriptase (Invitrogen). RT-qPCR in a 10-μL reaction using SYBR Green Mastermix (Bio-Rad) was run on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Primers (Supplemental Table S7) were designed as described previously (Lei et al., 2014). Arabidopsis UBQ10 (AT4G05320) served as an internal control. An ABI Prism 7900HT Sequence Detection System (Applied Biosystems) was used for running the RT-qPCR. Dissociation curves were examined for amplification specificity. The mean fold change in gene expression was calculated as described previously (Zhu-Salzman et al., 2003).