480 sybr green 1

Total RNA was isolated using a standard TriReagent protocol and treated with DNAse (TURBO DNA-free, AM1907M, Ambion). An aliquot was used for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Applied Biosystems, UK) containing Oligo-dT and random primers. Real-time PCR was performed using Roche Fast Start SYBR Green I on a Lightcycler2 with Lightcycler 3.5 software or using Roche 480 SYBR Green I on a Lightcycler480II with Lightcycler 1.5.62 software. DNA amplification was for 35 cycles with an initial 10 min at 95 °C followed by 10 s at 95 °C, 6 s at 55 °C, and 14 s at 72 °C. Primers were used at 0.5 μmole/L. Sequences of PCR primer are specified in Table 1. The specificity of PCR was verified by reactions without RT (-RT) and by melt-curve analysis. PCR cycle crossing-points (CP) were determined by fit-points methodology. Relative abundance of target RNA was calculated from (E_18S^Cp)/(E_target^Cp). PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. All quantitative PCR reactions were performed in duplicate and the data averaged to generate one value per experiment. On Fig. 1D, data are presented as Relative abundance. On Figs 2–6, data are presented after normalization to the Control group mean value.

Total RNA was isolated using a standard TriReagent protocol and treated with DNAse (TURBO DNA-free, AM1907M, Ambion). An aliquot was used for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Applied Biosystems, UK) containing Oligo-dT and random primers. Real-time PCR was performed using Roche Fast Start SYBR Green I on a Lightcycler2 with Lightcycler 3.5 software or using Roche 480 SYBR Green I on a Lightcycler480II with Lightcycler 1.5.62 software. DNA amplification was for 35 cycles with an initial 10 min at 95 °C followed by 10 s at 95 °C, 6 s at 55 °C and 14 s at 72 °C. Primers were used at 0.5 μM. Sequences of PCR primers are specified in Supplementary Table 1. The specificity of PCR was verified by reactions without RT (-RT) and by melt-curve analysis. PCR cycle crossing-points (CP) were determined by fit-points methodology. Relative abundance of target RNA was calculated from (E18sCp)/(EtargetCp). All quantitative PCR reactions were performed in duplicate and the data averaged to generate one value per experiment.

RNA was extracted from 3 × 10⁵ MDDCs using TRIZOL^® RNA isolation protocol for gene expression quantification. Real time quantitative PCR (qPCR) was performed after reverse transcription (RT) using a Roche LightCycler 480 with Roche 480 SYBR Green I master reagent according to manufacturer specifications. The relative abundance of each target mRNAs was calculated based on the standard curve and normalized using GAPDH as a control.
For qPCR, the primers used were the following:
TSPAN7 set 1 F 5′-CCTTCGTCTTCTGGATCACTGGGG-3′;
TSPAN7 set 1 R 5′-CATGGTCCACTGCCCGGCTC-3′;
TSPAN7 set 2 F 5′-CATCGCTGGAGTGGCGTTTGGA-3′;
TSPAN7 set 2 R 5′-TGCACGTTGTGGGGTAAGGGG-3′;
GFP F 5′- ACGTAAACGGCCACAAGTTC-3′;
GFP R 5′-AAGTCGTGCTGCTTCATGTG-3′.