Taqman reverse transcription reagents kit

Total RNAs were extracted from the collected cells and serum exosomes using TRIzol reagent (Invitrogen, USA). The concentration, integrity, and purity were measured by agarose gel electrophoresis and an UV spectrophotometer. Reverse transcription (Invitrogen, USA) was performed with a TaqMan Reverse Transcription Reagents kit, and SYBR _ PREMIX EXTAQ II (Takara, Dali, China) and ABI 7500PCR (Applied Biosystems, USA) were adopted for amplification. Amplification system: 10 μL of SYBR Premix Ex Taq II (2X), 2 μL of cDNA, 0.8 μL each of upstream primer and downstream primer, and supplemented to 20 μL with sterile purified water. Amplification conditions: pre-denaturation at 95° C for 30 s, denaturation at 95° C for 5 s, annealing and extension at 60° C for 30 s, for 40 cycles. Three replicate wells were set up for each sample and the experiment was repeatedly conducted 3 times. U6 was used as miR internal reference, and 2^-ΔΔCT was used for data analysis [35 (link)].

Total RNA was extracted from cells 48 h after transfection using Trizol reagent (Invitrogen). Trizol Reagen (600 μl) was added, and the samples were placed on ice for 20 min to lyse the cells. Subsequently, the cell lysate in Tirol was phase separated by addition of 120 μl of chloroform, and the RNA was precipitated by addition of 300 μl of isopropanol, and then, cDNA was synthesized by utilizing the TaqMan Reverse Transcription Reagents kit (TaKaRa). Real-time quantitative PCR (RT-qPCR) analysis was performed with different primer sequences using the SYBR Green Master Mix (TaKaRa). The primers used for RT-PCR were in Table 1.