Power sybr green chemistry

Arabidopsis seedlings were immediately frozen in liquid nitrogen, and stored at-80 °C. RNA was isolated using Trizol (Sangon) and reverse-transcribed using a reverse transcription kit (DRR047A) (Takara, http://www.takara-bio.com/). Quantitative RT-PCR was performed in a Bio-Rad CFX96 Real-time System (Bio-Rad, http://www.bio-rad.com) using Power SYBR green chemistry (DRR081A) (Takara). Primer sequences used are listed in S2 Appendix.
For CK-induced type-A ARR expression, seeds were germinated on MS plates and grown for 7 days. Treatments were carried out by incubating seedlings in liquid 1/2 MS culture medium containing 1% sucrose and supplemented with 10 μM tZ for 30 min [45 (link)].
For CK-induced GFC1 expression, we treated seedlings with 1 μM tZ for 30 min.

Total RNA of mutant or wild type was isolated using RNA kit (Tiangen, Beijing,
China) following manufacturer’s instructions. The concentration and quality of
RNA were verified by absorption 260/280 nm ratio between 1.8 and 2.0 using
Nanodrop2000 spectrophotometer (Thermo Scientific, USA). RNA quality was checked
on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and RNA integrity number
(PRIN) values were greater than 8.6 for mutant and wild type.
Total RNA from 23-19 wild type and eibi1 mutant were treated
with DNase and reverse-transcribed using a reverse transcription kit (RR047A)
(Takara Biomedical Technology, Beijing, China). Quantitative RT-PCR was
performed in an Agilent Stratagene MX3000P (Agilent) using Power SYBR green
chemistry (RR820A) (Takara). All qRT-PCR reactions were performed in triplicate
following the reaction conditions: 95 °C for 10 min followed by 40 cycles of 95
°C for 10 s and 60 °C for 31 s, and the results were analyzed with the relative
quantification system based on the 2^-ΔΔCt method. Actin primers were
5’-AAGTACAGTGTCTGGATTGGAGGG-3’ (sense) and 5’-TCGCAACTTAGAAGCACTTCCG-3’
(antisense).

Arabidopsis seedlings were immediately frozen in liquid nitrogen, and stored at −80 °C. RNA was isolated using Trizol (Sangon) and reverse-transcribed using a reverse transcription kit (DRR047A) (Takara, http://www.takara-bio.com/). Quantitative RT-PCR was performed using a Bio-Rad CFX96^TM Real-time System (Bio-Rad, http://www.bio-rad.com) and Power SYBR green chemistry (DRR081A) (Takara), with primers listed in Supplementary Appendix S1.
CK- and auxin-inducible gene expression was analyzed in 7-day-old seedlings grown on MS medium, as previously described20 (link), using treatments consisting of exposure to 10 μM tZ for 30 min51 (link) or 20 μM IAA for 1.5 h52 (link) in liquid MS medium. There were four independent biological replicates per treatment.