Evo m mlv rt kit with gdna clean for qpcr 2 ag11711

Three-month-old somatic embryo seedlings were cultured in an incubator under white light (16 h light, 8 h dark) on 3/4MS medium. They were treated separately with 100 mg/L abscisic acid (ABA), 100 mg L⁻¹ GA, 100 mg L⁻¹ chlormequat (CCC), 20% polyethylene-glycol (PEG)-6000 solution, 100 mM NaCl solution, or low temperature (4 °C). Mature leaves, stems, and roots were sampled from three biological replicates of the treatment and control plants at 0, 6, 12, 24, and 48 h after the trial. Quantitative RT-PCR analysis was used to confirm the expression patterns of LcGA2ox and LcGA20ox in the different organs under the different treatments (Table S4). Total RNA extraction was performed using a KK Fast Plant Total RNA Kit. First-strand cDNA was synthesized from 1.0 mg of RNA with an Evo M-MLV RT Kit with Gdna Clean for qPCRII AG11711 (Accurate Biotechnology (Hunan) Co., Ltd.). The qRT-PCR was carried out using SYBR-green fluorescence in a Roche LightCycler^®480 Real-Time PCR System. The ΔΔCT method was used to calculate the gene relative expression levels [44 (link)]. All qRT-PCR primers were designed by Primer5.0 and are listed in Table S5.

Total RNAs were extracted using the KK Fast Plant Total RNA Kit (ZOMANBIO, Beijing, China). The Evo M-MLV RT Kit with gDNA Clean for qPCRII AG11711 (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) was applied to synthesize the first-strand cDNA from 1.0 mg RNA. Equalbit 1× dsDNA HS Assay Kit (EQ121-01, vazyme) completed quantification of all reversed cDNAs. PCR amplifications were carried out with SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) in 20 μL volumes using Roche LightCycler^® 480 Real-Time PCR System. Three replicates were performed for each selected gene. The 18S was taken as a reference gene. The relative expression levels were calculated by the ∆∆ CT method [80 (link)]. All qRT-PCR primers were designed by Primer5.0 [81 ] and are listed in Table S5.

Total RNA was extracted with TRIzol Reagent (ambin, United States). After gDNA was removed, 500 ng of total RNA was reversed with Evo M-MLV RT Kit with gDNA Clean for qPCR II AG11711 (Accurate Biology) according to the manufacturer’s instructions. Real-time PCR was performed using PerfectStart Green qPCR SuperMix (TransGen Biotech) on a LightCycler 96 Detection System (Roche). GAPDH was served as an internal reference gene, and the primers used for qPCR in this study were as follows: MALT1: F, TGG​AAG​CCC​TAT​TCC​TCA​CTA​CC; R, CAT​GAC​ACC​AG-TAG​GTT​CCT​TGG, GAPDH: F, GAT​ATT​GTT​GCC​ATC​AAT​GAC​C; R, AGC​C-TTC​TCC​ATG​GTG​GTG​AAG​A, miR-218-5p: F, TTG​TGC​TTG​ATC​TAA​CCA​TGT; miR-338-3p: F, TCC​AGC​ATC​AGT​GAT​TTT​GTT​G; miR-365-3p: F, TAA​TG-CCC​CTA​AAA​ATC​CTT​AT; miR-375-3p: F, TTT​GTT​CGT​TCG​GCT​CGC​GTG​A; and U6: F, CGT​TCA​CGA​ATT​TGC​GTG​TCA​T. The reverse primer used in the qPCR of miRNA was the mRQ 3′ primer supplied with the microRNA first-strand synthesis and miRNA quantitation kits (Takara).