Primers and probes

This study adopted the prototype Fully Automated Nucleic Acid Amplification Testing System (FANAT-1), as well as primers and probes (Sangon Biotech) and nucleic acid extraction kit (QIAGEN). The basic PCR system contains High-Affinity HotStart Taq and TIANSeq M-MLV (defined as N enzyme, TIANGEN Biotech, Cat nos. ET108 and NG212, separately), MgCl₂ (25 mM) (Sangon Biotech, Cat no. B601193), One-Step PrimeScript III (defined as A enzyme, Takara Biotech, Cat no. RR601A), Anstart One-Step RT-PCR Mix (heat-labile UDG) (defined as E enzyme, Fapon Biotech, Cat no. MD013P), and 5 × Neoscript RT Premix-UNG (Probe qRT-PCR) (DG) (defined as F enzyme, Biori Biotech, Cat no. FM5254).

Total cellular RNAs were isolated from DCs using AxyPrep Multisource Total RNA Miniprep Kit (Axygen), and RNA samples were transformed into complementary DNA (cDNA) using RevertAid First-strand cDNA Synthesis kit (Thermo) according to the manufacturer's instructions. Quantitative PCR was performed on selected targets (mTOR, P70S6K, and 4EBP1) using pre-developed primers and probes (Sangon Biotech) on the QuantStudio (TM) real-time PCR software System. β-actin was used as an internal control. Primer sequences were the following: β-ACTIN, 5′-CCT GGC ACC CAG CAC AAT-3′ and 5′ -GGG CCG GAC TCG TCA TAC-3′; mTOR, 5′-ACT GGA GGC TGA TGG ACA CA-3′ and 5′-GGC TCT CCA AGT TCC ACA CC-3′; P70S6K, 5′-CAT CGG CAC CAC TTC CAA TA-3′ and 5′-TTC ATA CGC AGG TGC TCT GG-3′; 4EBP1, 5′-TCG GAA CTC ACC TGT GAC CA-3′ and 5′-GCT CAT CAC TGG AAG GGC TG-3′. Expression of mTOR, P70S6K, and 4EBP1 were calculated as described by the manufacturer.