Sybr green pcr mastermix

All qPCR assays were performed as described in Barbau-Piednoir et al., 2010 and Fraiture et al., 2014 using the primers indicated in Table 1 [6 (link), 14 (link)]. More precisely, a standard 25 μl reaction volume was applied containing 1X SYBR®Green PCR Mastermix (Diagenode, Liège, Belgium), 250 nM of each primer and 5 μl of DNA (10 ng/μl). The qPCR cycling program consisted of a single cycle of DNA polymerase activation for 10 min at 95 °C followed by 40 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing-extension step). The program for melting curve analysis was performed by gradually increasing the temperature from 60 to 95 °C in 20 min (±0.6 °/20 s). All runs were performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK). For each assay, a “No Template Control” (NTC) was included.

For the real-time PCR screening, 25 ng of DNA from each sample were tested using the p35S, pNOS, tNOS, t35S pCAMBIA, Cry1Ab/Ac, t35S, A1, CAP and CRT2 markers (Table 1). The real-time PCR mix contained 1X SYBR®Green PCR Mastermix (Diagenode) and 250 nM of each primer in a total volume of 25 µl. The real-time PCR program consisted of initial DNA polymerase activation for 10 min at 95 °C followed by 40 amplification cycles of 15 sec at 95 °C (denaturing step) and 1 min at 60 °C, for the p35S, pNOS, tNOS, t35S pCAMBIA, Cry1Ab/Ac, t35S, CAP and CRT2 markers, or 65 °C, for the A1 marker, (annealing-extension step). Melting curve analyses were performed by gradually increasing the temperature from 60 to 95 °C in 20 min (±0.6°/20 sec). All runs were performed on an ABI 7300 real-time PCR system (Life Technologies). For each assay, a “No Template Control” (NTC) was included.

Briefly, 2 µg of total RNA were treated with RNase-free DNase I (Promega) and reverse transcribed using the iScript kit (Bio-Rad, Irvine, CA, USA) according to the manufacturer`s instructions. Two µl of cDNA served as a template in a 20 µl qPCR reaction mix containing the primers and SYBR Green PCR Master Mix (Diagenode, Seraing, Belgium). Quantification of gene expression was performed using the ABI PRISM^® 7000 SD RT-PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). A relative standard curve was constructed with serial dilutions (1:1, 1:10, 1:100) using a pool of the cDNA generated from all animals used in the study. The amplification program consisted of 1 cycle of 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec, annealing for 10 sec, and 72°C for 30 sec. The fluorescent intensity was measured at a specific acquisition temperature for each gene. Data were extracted and amplification plots generated with ABI SDS software (Applied Biosystems, Thermo Fisher Scientific). All amplifications were performed in duplicate, and C_t scores were averaged for subsequent calculations of relative expression values. The level of individual mRNA was normalized to the level of the housekeeping gene cyclophilin by using the Pfaffl method (42 (link)). Exon-specific primers (Table S1) were designed by the Primer 3 program (43 (link)).