Abi 7500 real time pcr system thermocycler

Real-Time PCR primers for target mRNAs were designed using the Primer-BLAST tool (30 (link)) based on the reference species (Table 1). The real-time PCR assay was carried out in the ABI 7500 Real-Time PCR system thermocycler (Applied Biosystems; USA) in singleplex mode. Each PCR reaction tube contained 10 μL of the FastStart SYBR Green Master ROX mix (Roche Diagnostics), 0.25 (CYP1A1) or 0.5 (GSTP1) μM of each primer (forward and reverse; Table 1), 1 μL of previously synthesized cDNA as a template supplemented with PCR-grade H₂O to a final volume of 20 μL. Further details of the analytical procedure have been described elsewhere (31 (link)).

To assess the plant defense mechanisms induced by the exogenous foliar application of PeBb1 elicitor on B. rapa plants, relative expression of key genes associated with B. rapa’s ET and JA pathways (Table 2) were examined by real-time quantitative PCR (RT-qPCR). Using the EasyPure^® Plant RNA Kit (TransGen Biotech, Beijing, China), total RNA was extracted from the leaves of aphid infested protein-treated and buffer-treated (control) plants of B. rapa. TransScript^® All-in-One SuperMix for qPCR Kit (TransGen Biotech, Beijing, China) was used to synthesize first-strand cDNA. The relative expression levels of both type of defense-related genes were determined by RT-qPCR performed on ABI 7500 Real-Time PCR System thermocycler (Applied Biosystems, Foster City, CA, USA) using TransStart^® Green qPCR SuperMix UDG (TransGen Biotech, Beijing, China). Three independent biological and three technical replicates were performed for each sample. Actin was used as a quantitative control. Relative expression levels were calculated using the 2^–ΔΔCT method [44 (link)]. Primer pairs used for the amplification of these plant defense associated genes are detailed in Table 3.

Molecular confirmation of DENV infection was performed by using US CDC DENV-1-4 real-time RT-PCR Multiplex Assay (CDC, Atlanta, USA). The kit was obtained from the US CDC Dengue Branch. The assay was performed in multiplex on ABI 7500 real-time PCR System thermocycler (Applied Biosystems, Foster City, CA, USA), as described in the package insert.