Identification of novel diagnostic markers for fatal hypothermia by body temperature-dependent gene expression analysis in rats

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice. In this study, in order to identify novel molecular markers of fatal hypothermia, we made rat models of mild, moderate and severe hypothermia, and performed body temperature-dependent gene expression analysis in illiopsoas muscle using next-generation sequencing. Twelve male wistar rats (Pathogen-free 9-week-old) were divided into 4 groups (control, mild, moderate and severe hypothermia), which were belonged 3 rats, and were anesthetized with intraperitoneal. After 30 minutes, they were treated at cold (ambient temperature: 4 °C), and chronologically measured rectal temperature (Ret). Control was euthanized by cervical dislocation at 30 minutes after anesthesia. Mild hypothermia was done by cervical dislocation when the Ret reached 30°C. Similarly, moderate and severe hypothermia also was done when the Ret did 22°C and 12°C, respectively. Total RNA from iliopsoas muscle was extracted using RNeasy Protect Mini kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. RNA integrity was assessed by on-chip capillary electrophoresis using an Agilent RNA 6000 Nano kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). NGS was performed using 2000 ng of total RNA. Three libraries were generated for each of the four conditions (Control, mild, moderate and severe hypothermia) (TruSeq Stranded mRNA LT Sample Prep Kit, Illumina, San Diego, California, USA), and sequenced on an Illumina Miseq.