Thermal cycler dice real time system single mrq

Total RNA was extracted from astrocytes in each well of a 24-well plate using ISOGEN^® (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Purified total RNA was used to synthesize cDNA with a PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) for 15 min at 37°C and for 5 s at 85°C. cDNA was amplified in a PCR reaction containing SYBR^® Premix Ex Taq^® (Takara Bio) and 0.4 μM of each gene-specific primer (Table 1). The reactions were performed in a Thermal Cycler Dice Real Time System Single MRQ (Takara Bio; TP870) with a PCR profile comprising an initial denaturation step for 2 min at 95°C, 40 cycles with 5 s at 95°C and 60 s at 60°C, and a final step for 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C. Following amplification, reaction specificity for each PCR run was confirmed by melt curve analysis.

qRT-PCR experiments to quantify egfp and Sh ble transcripts in the transformed cells were performed in triplicate on a Thermal Cycler Dice^® Real Time System Single MRQ (Takara Bio Inc., Otsu, Japan) using 2 μl of the cDNA mixture added as a template and SYBR^®Premix Ex Taq^TM II (Tli RNaseH Plus) (Takara Bio Inc., Otsu, Japan). The cycling conditions involved denaturing for 30 s at 95 °C and 40 cycles of melting (5 s at 95 °C) and annealing coupled with an extension (30 s at 60 °C). To determine promoter activities, each qRT-PCR measurement was performed in triplicate using primers to identify the abundance of Sh ble mRNA and egfp mRNA (shown in Supplementary Table 1). The egfp mRNA levels were normalized by dividing by the Sh ble mRNA levels to minimize variations in transgene expression due to multiple insertions (Fig. 1b).

Total RNA from pDCs (10⁶) was extracted with TRIzol (Life Technologies), and cDNA was synthesized with oligo (dT)₂₀ as a primer using the PrimeScript™ RT Reagent Kit (Takara). Transcriptional expression levels were measured by real-time PCR (Thermal Cycler Dice Real Time System Single MRQ, Takara) using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara) with a pair of specific primers for Siglech (5′-aat tca cag aac tcc aca gc-3′ and 5′-gat ccc aag aag cag gaa tt-3′), Ifna (5′-tct gat gca gca ggt ggg-3′ and 5′-agg gct ctc cag act tct gct ctg-3′), Infb (5′-gca ctg ggt gga atg aga ct-3′ and 5′′-agt gga gag cag ttg agg aca-3′), Mx1 (5′-gat ccg act tca ctt cca gat gg-3′ and 5′-cat ctc agt ggt agt caa ccc-3′) and Gapdh (5′-aaa ttc aac ggc aca gtc aag-3′ and 5′-tgg tgg tga aga cac cag tag-3′) after normalization for the expression of Gapdh.