Luminaris higreen qrt pcr master mix

Twenty-one representative BL-responsive genes (VC0837, VC0943, VC1118, VC1248, VC1263, VC1359, VC1392, VC1484, VC1570, VC1643, VC1814, VC1922, VC2088, VC2301, VCA0055, VCA0615, VCA0782, VCA0798, VCA0809, VCA0957, VCA1087) were selected for validation by qRT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After DNase I treatment, 1 μg total RNA was used for first-strand cDNA synthesis using random hexamer oligos. The qRT-PCRs were performed with Luminaris HiGreen qRT-PCR Master Mix (Thermo Scientific, Waltham, MA, USA) on the CFX Connect™ Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA) using gene-specific primers (Supplementary Table S10), with gapdh (VC2000) as the internal reference gene. The amplification program was as follows: 95 °C for 10 min; 40 cycles of 95 °C for 2 s, 56 °C for 10 s, and 72 °C for 10 s, followed by a thermal denaturing step to generate the melting curves. All reactions were performed in biological triplicate (each triplicate with two technical replicates), and the results were expressed relative to the transcript level of gapdh in each sample using the 2^−ΔΔCT method61 (link). The mRNA expression data were analyzed using IBM SPSS ver. 20.0 (SPSS Inc., Chicago, IL, USA). All relative mRNA expression data are presented as mean ± S.D. (n = 6).