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RNA extraction, cDNA synthesis, and qRT-PCR

SL Sanghwa Lee
WW Wenli Wang
EH Enamul Huq
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Whole seedlings, shoot and root tissues from 6-day-old white light-grown seedlings were used with three independent biological replicates (n = 3) for RNA extraction. Seedlings were grown at 22 °C under continuous white light for 6 days and then were either kept at 22 °C or transferred to 28 °C for additional 4 h under continuous white light before samples were harvested. Plant RNA purification kit from Sigma-Aldrich were used for total RNA extraction following the manufacturer’s protocols. For cDNA synthesis, 1 μg of total RNA was used for reverse transcription with M-MLV Reverse Transcriptase (Thermo Fisher Scientific). SYBR Green PCR master mix (Thermo Fisher Scientific) and gene-specific oligonucleotides were used to conduct qPCR analyses. Finally, relative transcription level was calculated using 2ΔCt with ACT7 as a control.

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