Cfx96 real time system c1000

For RT-qPCR, plant total RNA was extracted using Ambion PureLink RNA Mini Kit (Invitrogen, 12183018 A). The first-strand cDNA was then synthesized from 2 μg of the extracted total RNA using anchored oligodT₁₈VN and ProtoScript II (NEB, M0368) reverse transcriptase. For McrBC-qPCR, plant genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. 100 ng of genomic DNA was then digested with the methylation-dependent restriction endonuclease McrBC (NEB, M0272L) for 7.5 h at 37 °C followed by 65 °C for 20 min to deactivate the enzyme. The quantitative real-time PCR was performed in triplicates using SYBR Green qPCR Master Mix (Bio-Rad, 1725124) and a Bio-Rad CFX96 C1000 Real-Time system. The gene expression levels in RT-qPCR were normalized against wild-type control and internal control ACT7. The relative methylation levels of McrBC-qPCR were normalized to uncut control. The primer sequences are listed in Supplementary Table 3.

MiRNA expression levels were quantitated using a SYBR Primescript miRNA RT PCR Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions with a Bio-Rad CFX™ 96 C1000 Real-Time system. Briefly, the RNA samples were converted to cDNA by a One-step Primescript miRNAcDNA Synthesis Kit (TaKaRa, Dalian, China), followed by real-time qPCR and normalized using U6 small nuclear RNA (RNU6B) by the 2^−ΔCT method. Primers for miR-214 and U6 were from GeneCopoeia (HmiRQP0320, HmiRQP9001). All reactions were run in duplicate.

RNA samples were reverse transcribed to cDNA using reverse transcriptase cDNA synthesis kit (Toyobo, Osaka, Japan). We detected the expression levels of PRSS22 messenger RNA (mRNA) by qPCR using SYBR Green Real-time PCR Master Mix (Roche, Mannheim, Germany) on Bio-Rad CFX™ 96 C1000 Real-Time system. The relative expression of target gene was normalized to GAPDH expression, which yielded the 2^−ΔCt value.

To analyze the gene expression, the cells (0.5 × 10⁵) were seeded in 6-well plates and incubated with 1-µM etoposide and/or 10–50-µg/mL kaempferol, P2, P5, and P7 at 37 °C for 24 h. Afterwards, the cells were collected, and the total RNA from each sample was extracted using the Universal RNA Purification Kit (EurX, Gdansk, Poland), according to the manufacturer’s instructions. Reverse transcription and real-time PCR reactions were performed using the SensiFAST™ Probe No-ROX One-Step Kit (Bioline, London, UK) on the CFX96 C1000 real-time system (Bio-Rad, Hercules, CA, USA). The relative expression was evaluated using the TaqMan gene expression assay for HO-1: Hs00157965_m1, NFE2L2: Hs00975961_m1, NQO1: Hs00168547_m1, SOD1: Hs00533490_m1, SOD2: Hs00167309_m1, and GAPDH: Hs02786624_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The GAPDH gene was utilized as a reference gene. Each assay was performed in quadruplicate. The relative gene expression was calculated as the fold change according to the control sample based on the double-delta Ct method.

For mRNA quantitative analysis, total RNAs were prepared using Trizol (Invitrogen) and were reverse transcribed with a Rever Tra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was performed in a total volume of 10-μl SYBR Green Real-time PCR Master Mix (Roche, Mannheim, Germany) on a Bio-Rad CFX™ 96 C1000 Real-Time system. For quantitative detection of miRNA, reverse transcription and qPCR assay were performed using the All-in-One™ miRNA qRT-PCR detection kit (Genecopeia, Rockville, MD, USA). Primers for miR-148a-3p (HmiRQP0204), miR-148b-3p (HmiRQP0206), miR-152-3p (HmiRQP0213) and U6 (RNU6B, HmiRQP9001; reference gene) were from GeneCopoeia.