Transient plant production of Salmonella Typhimurium diagnostic antibodies

Kopertekh, Lilya; Meyer, Torsten; Freyer, Cornelia; Hust, Michael

RNA for qPCR analysis was isolated from agroinfiltrated N. benthamiana leaves taken at 5 dpi using the RNeasy Mini Kit from Qiagen (Qiagen) according to manufacturer's specifications. The first strand cDNA was synthesized using Maxima Reverse Transcriptase and random hexamer primer with 2 mg of total RNA in a reaction final volume of 20 ml following manufacturer instructions (Thermo Scientific). Real-time PCR reactions were performed with Mastercycler 1 ep realplex (Eppendorf). Reaction mixture contained 2 ml of cDNA, 10 ml of 2x Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and 0.8 ml of 10 mM primers in a total volume of 20 ml. Primer sequences for the TM43-E10 and reference cyclophilin (cyp) genes are listed in Table S1 . PCR thermal cycles were as follows: initial denaturation step for 10 min at 95 C followed by 45 cycles of denaturation for 15 s at 95 C, annealing for 30 s at 60 C and extention for 30 s at 72 C. Relative quantifications were performed based on the DDCT method [36] using cyp gene as an internal standard. Three biological replicates and three technical replicates for each sample were analysed by qPCR.