Rna 6000 nano gel system

Peripheral whole blood (20 ml) was collected at AM 6:00 to 8:00 after written informed consent was obtained from all the recruited participants. The PBMCs were isolated by Ficoll-Hypaque gradient centrifugation (HISTOPAQUE^®-119, Sigma-Aldrich, Inc., St. Louis, MO USA) within 90 min of drawing blood, washed in PBS, and then stored in RNAlater (Ambion Inc., Austin, TX, USA) at -80°C until RNA isolation. An RNeasy^® Plus Mini Kit (Qiagen, Hilden, Germany) was used for isolation of high quality total RNA, and treated with DNase according to the manufacture protocol. RNA samples were run on a RNA 6000 Nano Gel System (Agilent Technologies Inc., Palo Alto, CA, USA) using an Agilent 2100 Bioanalyzer (Agilent) to determine the quality of RNA. Only samples with A260/A280 ratios of 1.9 to 2.1 were used for further analysis. A total of 300 ng RNA was used for synthesis of first strand cDNA and transcription of cRNA using an Illumina Totalprep RNA Amplication kit (Ambion, Inc.).

Fresh venous blood 20 mL was collected from each participant on awakening and immediately transferred to a tube containing 3.2% sodium citrate (1:10 dilution). PBMCs were isolated by Ficoll-Hypaque gradient centrifugation (HISTOPAQUE^®-119, Sigma-Aldrich, Inc., Burlington, MA, USA), and then stored in RNAlater (Ambion Inc., Austin, TX, USA) at −80 °C until RNA isolation. A miRNeasy Mini Kit column-based system (Qiagen, Valencia, CA, USA) was used for isolation of total RNA, and treated with DNase. RNA samples were run on an RNA 6000 Nano Gel System (Agilent Technologies Inc., Santa Clara, CA, USA) using an Agilent 2100 Bioanalyzer (Agilent) to determine the quality of RNA. Only samples with A260/A280 ratios of 1.9 to 2.1 and RNA integrity number ≥ 8 were used for further analysis.