Tb green premix ex taq 2 tli rnase h plus master mix

RT-qPCR validation was performed on 18 dysregulated genes (Table 1) using total RNA samples subjected to RNA-seq. Reverse transcription was performed using 500 ng total RNA using PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) according to the manufacturer’s instructions. Equal amounts of cDNA (1 μl) were assessed in total volumes of 20 μl containing TB Green Premix Ex Taq II (Tli RNaseH Plus) Mastermix (Takara Bio Europe, France) and primers (200 nM) (Table 1). The mixtures were incubated at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, and 60°C for 50 s. The reactions were carried out in duplicate using a Rotorgene Q (Qiagen). The fold changes were calculated by relative quantification using the ΔΔCt method [21 (link)]. The data were normalized using HPRT and GAPDH as reference genes, and the relative gene expression was set to 1 for the control (non-infected) samples.

Total RNA was extracted from the anticoagulated blood samples with the TaKaRa RNAiso Plus Extraction Kit (Takara Bio, Inc., Kusatsu, Japan), according to the manufacturer’s instructions. SVA RNA was detected with qRT–PCR targeting the SVA L/VP4 polymerase gene, and the limit of detection was 64 copies/μL, as previously described [38 (link)]. Briefly, reaction was set in a 25-μL volume containing 12.5 μL of 2 × TB Green Premix Ex Taq II (Tli RNaseH Plus) Master Mix (Takara Bio, Inc.), 2 μL of cDNA or DNA template, 8.5 μL of diethylpyrocarbonate (DEPC)-treated water and 1 μL (the stock concentration 10 μM) of each primer (SVA-FP1 (5′-TGAAGTTGCGGAGAAGAT-3′) (location: 878–896) and SVA-RP1 (5′-TTGCGTAGTAATTGAAGGT-3′) (location: 966–985) ( GenBank accession no. MN922286)). Amplification conditions included an initial denaturation step at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. Amplification was performed using the CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). Melting curve analysis was performed using Bio-Rad CFX96 software.