2.6. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis of Clock Genes
BLOOD, SHIGA, AMPLIFICATION
Gene expression was analyzed using peripheral blood mononuclear cells (PBMCs). Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes. PBMCs were isolated by density gradient centrifugation at 3000 rpm for 30 min in Ficoll–Paque medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Total RNA was extracted using the TRI reagent, and cDNA was synthesized with 1 μg of total RNA using an RT-PCR Kit (Takara Bio Inc., Shiga, Japan). PCR was performed using a Thermal Cycler Dice Real-Time System (Takara Bio Inc., Shiga, Japan). Amplification was performed under the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. Relative gene expression was analyzed using the comparative Ct method. Gene expression was determined using the 2