Cfx connect real time pcr detection system device

Leaves from five plants per biological replication were collected and fixed in liquid nitrogen 1 and 3 days after population with aphids. Total wheat RNA was extracted using TRIzol™ Reagent (Merck KGaA, Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s instructions. cDNA synthesis was carried out as described previously [33 (link)]. Primers for qRT-PCR were designed using a web-based primer designing tool from IDT (http://eu.idtdna.com/Scitools/Applications/Primerquest, accessed on 10 November 2022) (USA). The sequences of all the primers are presented in Table S2 (Supplementary Materials). Quantitative PCR was performed by polymerase chain reaction in real time using a set of predefined reagents EvaGreenI (Synthol, Moscow, Russia) and CFX Connect real-time PCR Detection System device (BioRad Laboratories, Hercules, CA, USA). To standardize the data, wheat gene TaRLI (RNaseLinhibitor-like) (Table S2, Supplementary Materials) was used as an internal reference for the real-time qPCR analysis. The quantification of gene expression was performed using CFX Connect real-time PCR Detection System (BioRad Laboratories, Hercules, CA, USA). In order to quantify the relative gene expression using the delta-delta Ct method was performed as described earlier [33 (link)]. Three independent biological and three technical replications were performed for each experiment.

For viral diagnosis, test samples were selected from healthy and diseased tomato plants at 7 and 14 days after being infected with PVX and PVY and fixed in liquid nitrogen. The pool of five plants per replicate (three leaves per plant) was prepared for qRT-PCR analysis. Total RNA was extracted using TRIzol™ Reagent (Merck KGaA, Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s instructions. The qRT-PCR was performed using specific primers for PVX CP (Coat Protein) and PVY PIPO (Pretty Interesting Potyviridae open reading frames(ORF)) genes by using the kit “Potato Virus X and Potato Virus Y” (Synthol, Moscow, Russia), according to the manufacturer’s protocols, on a CFX Connect real-time PCR Detection System device (BioRad Laboratories, Hercules, CA, USA). The housekeeping gene SlACT was used for the normalization of PVX CP and PVY PIPO expression in S. lycopersicum. Three independent biological replicates were performed for each experiment.