Taqman micro rna transcription kits

Venous blood (15 mL) samples were drawn between 6:00 a.m. and 10:00 a.m., after subjects fasted for 9 h. PBMCs were isolated from venous blood samples by Ficoll–Paque (GE, #17-5442-02) density gradient centrifugation and labeled with BD IMag anti-human CD14 Magnetic Particles DM (BD Biosciences, #557769) according to the manufacturer’s directions. Labeled monocytes were collected for further analysis. All samples were stored at −80 °C until assayed. RNA samples were extracted using Quick-RNA MiniPrep kits (Zymo Research R1055) and reverse-transcribed to complementary DNA (cDNA) using TaqMan Micro RNA Transcription Kits (Applied Biosystems 4366596). Specific sequences were amplified in an Applied Biosystems 7500 system using TaqMan^® Universal Master Mix II, without UNG (Applied Biosystems 4440040). The amplification conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 6 °C for 1 min. Gene expression was quantified using the software provided by the manufacturer (Applied Biosystems), analyzed according to the △△Ct method, and normalized to the expression of U6 small nuclear RNA (snRNA). Primers for qRT-PCR are showed in Table S1 (Supplementary Materials).

Total RNA was extracted from the serum exosomes using the SeraMir Exosome RNA Amplification kit (System Biosciences, Cat#RA806A-1) according to the manufacturer′s protocol, and reverse-transcribed to complementary DNA (cDNA) using the TaqMan Micro RNA Transcription Kits (Applied Biosystems 4366596, Foster City, CA, USA). The thermal cycling conditions are 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min.