Lightcycler 480 sybr 1 master pcr mix

Leaves were harvested from plants infected with TSWV or infiltrated with A. tumefaciens cultures harboring different constructs, and ground in liquid nitrogen. Total RNA was extracted from leaves, and first-strand cDNA was synthesized using PrimeScript First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). RT-PCR was used to check NSm gene expression following inoculation with TSWV. Plants with no obvious symptoms and no NSm expression were classified as resistant, and the others were designated as susceptible.
A qRT-PCR assay was performed on the LightCycler 480^@ II machine with LightCycler 480^@ SYBR I Master PCR Mix (Roche Applied Science, Basel, Switzerland). Thermocycling conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s, and lastly, a melting curve analysis (68 °C to 95 °C). Primers used in the qRT-PCR assay were designed using the Premier 6 (Premier-Biosoft, Palo Alto, CA, USA) software (Table A1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control to normalize gene expression data. Six biological and three technical replicates were performed for each experiment, to minimize biological variation and random noise associated with the equipment.

To confirm the results of transcriptome analysis, the expression levels of 28 selected genes were measured in systemic leaves of mock- and TZSV-infected N. tabacum using qRT-PCR. Total RNAs were extracted using TRIzol reagent (Invitrogen) and treated with DNase I (TaKaRa, Dalian, China) to remove residual genome DNA. The concentration of total RNA was adjusted to 1 μg/μl with nuclease-free water and first-strand cDNA was synthsized using a PrimeScript First Strand cDNA Synthesis Kit (TaKaRa). qRT-PCR assay was performed on the LightCycler 480^@ II machine with LightCycler 480^@ SYBR I Master PCR mix (Roche Applied Science, Basel, Switzerland). Thermocycling conditions were as follows: 95 °C for 5 min; and 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s, followed by melting curve generation (68 °C to 95 °C). Primers used in qRT-PCR assay were designed using the Premier 6 (PremierBiosoft, Palo Alto, CA) software and shown in Table S1 (Additional file 1: Table S1). To normalize the amount of cDNA in each reaction during qRT-PCR, an internal control gene primers that target the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used with above PCR reagents and conditions. Three biological and three technical replicates were used for each experiment to limit the effect of biological variation and random noise associated with equipment.