Brilliant 3 sybr green qpcr master mix with low rox

Total RNA from apex leaves of WFP genotypes, from aphid-infected after 24 h and from healthy plants was extracted from frozen samples using the NucleoSpin^®RNA Plant Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s instructions. RNA yield and purity were quantified using a Nanodrop^® by spectrophotometric absorbance and agarose gel electrophoresis. In total, 500 mg of cDNAs were synthesized using the Affinity Script Multiple Temperature cDNA Synthesis Kit (Agilent, Santa Clara, CA, USA), following the instructions provided. Expression analysis was performed by quantitative real-time PCR (qRT-PCR) using a “Stratagen MX Pro 3005” and Brilliant III SYBR Green qPCR Master Mix with Low ROX (Agilent Technologies). The reaction was prepared with each primer (0.5 µL, 10 µM), 2 µL of 1:10 diluted cDNA, RNase-free water (5 µL), and Brilliant III SYBR Green qPCR Master Mix with High ROX (7 µL) in a total volume of 15 µL. PCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min. All qPCR reactions were normalized using the Ct value corresponding to the EiF4Ɣ peach gene (Pp_Eif4Ɣ) and the 60 S L13 ribosomal protein gene (Pp_RPL13). Each sample was measured three times with three technical replicates. The list of the designed primers for the qPCR is in Table S1.

hCMEC cultures were incubated with E. coli EV36, phage K1F or TNFα (Sino Biological, China) in biological triplicates. RNA was recovered using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) with DNase I Digestion Kit (Sigma-Aldrich) following the manufacturer’s protocol.
1 μg RNA samples were used for cDNA synthesis using 100 U Superscript III (Invitrogen) primed by 50 nM Random Hexamers (Invitrogen) and 10 μM dNTP Mix (Thermo Scientific).
Real-time PCR of cDNA samples was performed with Brilliant III SYBR Green qPCR Master Mix with Low ROX (Agilent) using the Stratagene Mx3005P instrument with MxPro v4.10 build 389 software (Agilent Technologies). Each sample was quantified in technical triplicate using primer sets for GAPDH^{67 (link)}, TNFα^{68 (link)}, IL-6^{69 (link)}, IL-8^{70 (link)}, IL-10^{71 (link)} and IFNβ^{72 (link)}.
Data analysis was performed using the Comparative Ct Method^{73 (link)}. ΔCt values for TNFα, IL-6, IL-8, IL-10 and IFNβ were calculated by subtracting the average GAPDH Ct value from the average Ct value obtained for each gene. ΔΔCt values were calculated by subtracting ΔCt values of untreated cultures from ΔCt values of treated cultures. The fold change was calculated by taking the log base 2 of ΔΔCt values.