Improm 2 reverse

TRI Reagent (BioScience Technology) was used to extract the total RNA from HaCaT cells as previously described [39 (link),40 (link),41 (link)]. According to the manufacturer’s instructions, RNA (1 μg) was reverse-transcribed using ImProm-II™ reverse transcriptase (Promega, Madison, WI, USA) to create cDNA. The cDNA levels of each target gene were measured by quantitative real-time PCR using the StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA) with FastStart Universal SYBR Green Master (Roche, Basel, Switzerland). The sequences of the forward and reverse primers of c-jun, egr-1, c-myc, c-fos, and β-actin were described previously [24 (link)].

Total RNA was isolated using the RNeasy Mini Kit. cDNA was synthesized with the ImProm-II Reverse (Promega, Madison, WI, USA). mRNA expression was carried out as described previously^{34 (link)} with ABI 7300 (Applied Biosystems, Foster, CA, USA) and SYBR Green PCR Master Mix (QPS-201, Toyobo, Japan). GAPDH (internal control) and PCR primers used in this study are listed in Table S2.

Louise genomic DNA was isolated from 100 mg of ground leaf tissue using the sodium bisulfite method [41 (link), 42 (link)]. Total RNA was extracted using the Trizol reagent (Invitrogen) method according to the manufacturer’s protocol. Before performing RT-qPCR, RNA samples were treated with 2 U of DNase I to prevent DNA contamination using the DNA-free RNA kit™ (Zymo Research). Nucleic acid concentration and quality were assessed by NanoDrop™ UV spectrophotometry and by agarose gel electrophoresis. For the cDNA cloning experiment, first-strand cDNA was synthesized from 5 μg of total RNA using the ImProm-II™ reverse transcription system kit (Promega) according to the manufacturer’s instructions. For the RT-qPCR experiment, first-strand cDNA was synthesized from 1 μg of total RNA using the ProtoScript® first strand cDNA synthesis kit (NEB) following the manufacturer’s instructions. Synthesized cDNA was adjusted to a final concentration of 100 ng/μl for cDNA cloning and to 2 ng/μl with RNase-free water for RT-qPCR experiments.