Chamq sybr qpcr master mix low rox premixed

Total RNA was isolated from rice or tobacco leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized using the fast quant RT kit (Vazyme, Nanjing, China). The resulting cDNA was used as the template for RT-PCR and RT-qPCR. RT-qPCR was conducted using the ChamQTM SYBR qPCR Master Mix (Low ROX Premixed) and ABI7900HT Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). The rice actin gene OsUBQ5 (AK061988) was used as an internal reference. The results were analyzed by the 2^-ΔΔCt method and shown as means ± SD (n = 3). At least three biological repeats were conducted for all experiments. The RT-qPCR primer sequences used are listed in Supplementary Table 1.

Total RNA was extracted from young trifoliate leaves using TRIzol reagent (Invitrogen, United States). About 1.5 μg of total RNA was reversely transcribed to cDNA using a reverse transcription kit (Tiangen Company, Beijing, China). Real-time PCR was conducted using ChamQ^TM SYBR qPCR Master Mix (Low ROX Premixed) by an ABI7900HT Sequence Detection System (Applied Biosystems, Carlsbad, CA, United States). The RT-qPCR conditions were as follows: 95°C for 4 min; 40 cycles of 95°C for 10 s and 60°C for 30 s. The relative expression levels of genes were determined using the 2^{–ΔΔC (t)} method (Livak and Schmittgen, 2001 (link)). The soybean Actin11 gene was used as an internal control. Three biological and two technical replicates were conducted to determine gene expression. The RT-qPCR primer sequences used in this study are listed in Supplementary Table 2.