Primescript plus rtase mix

Total RNA was extracted from 0.1 g liver sections using ISOGEN II (Nippon Gene, Tokyo, Japan). Quantitative RT-PCR was performed with the One Step SYBR PrimeScript PLUS RT-PCR kit (Perfect Real Time) (Takara Bio, Shiga, Japan) using an Applied Biosystems 7300 (Applied Biosystems, Foster City, CA). PCR was performed in a 20 μL of solution containing 0.4 μM primers, 0.4 μL ROX Dye, and sample RNA (30 ng) in 2X One Step SYBR RT-PCR Buffer 4, TaKaRa Ex Taq HS Mix and PrimeScript PLUS RTase Mix. PCR conditions were as follows: 42 °C for 5 min, 95 °C for 10 s, and 40 cycles of 95 °C for 5 s followed by 60 °C for 31 s. Primer pairs are shown in Table 1. Relative expression of each mRNA was determined using the standard curve method. The amount of each target mRNA quantified was normalized against that of GAPDH-encoding mRNA.

Total mRNA from stimulated Jurkat T cells was extracted using PureLink RNA Mini Kit (Life technologies). Relative expression of IL-2 was quantified by qPCR^{49 (link)}. Quantification of IL-2 mRNA was performed on a Thermal Cycler Dice Real Time System II (Takara) using the One Step SYBR PrimeScript PLUS RT-PCR kit (Takara). The primer pairs are shown in Table S8. The PCR mixture contained 2 μl of extracted mRNA, 10 μl of One Step SYBR RT-PCR buffer 4 (2x), 1.2 μl of Takara Ex Taq HS Mix, 0.4 μl of PrimeScript PLUS RTase Mix, primer pairs (optimized to final concentration of 10 μM) and sterile water to a final reaction volume of 20 μl. The optimal cycling conditions were 5 min at 42 °C, 10 s at 95 °C; 40 cycles of 5 s at 95 °C, 30 s at 60 °C. All qPCR reactions were performed in triplicate. Data were normalized against β-Actin expression, and the relative expression of IL-2 was calculated using the comparative C_T method.

The test of transcript variability among the fifteen samples (three organs and five stages) was carried out using qRT-PCR reactions for mRNA. These reactions were performed in triplicate using the MxPro 4.1 system assays and the One Step SYBR PrimeScript PLUS RT-PCR kit (TaKaRa, TaKaRa code: DRR096A), including minus reverse transcription (RT) controls to assess the genomic DNA and non-template controls, thereby ensuring a lack of background signal in the assay. The final volume of the RT reaction was 25 µl, which consisted of 12.5 µl 2×One Step SYBR RT-PCR Buffer, 1.5 µl TaKaRa Ex Taq HS Mix, 0.5 µl PrimeScript PLUS RTase Mix, 10 µM PCR Forward Primer, 10 µM PCR Reverse Primer, 40 ng total RNA, and 6.5 µl RNase-free H₂O. The reactions were incubated in thin-wall polypropylene 8-tube strips using MxPro 4.1. The PCR cycling conditions were as follows: 42°C for 5 min, 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Finally, the steps, 95°C for 15 sec, 60°C for 30 sec, and 95°C for 15 sec were carried out for dissociation. Data were collected during each cycle at the 60°C extension step.