Transcriptomic analysis of liver from mice subjected to simulated spaceflight euthanasia, freezing, and tissue preservation protocols

To understand the molecular mechanisms affected by spaceflight, it is essential to achieve high quality sample preservation on-orbit for downstream gene expression analysis. However, sample preservation protocols must also be compatible with available equipment and crew time. NASA's Rodent Research (RR) missions have used various methods for euthanasia, carcass preservation, and tissue preservation. This study extends the sample preservation study performed by GeneLab in GLDS-49 which examined conditions used for the RR-1 mission to include conditions used for multiple RR missions and is designed to help determine factors which may confound data analysis. To determine whether these various factors affect changes in gene expression, this ground-based study generated gene expression profiles measured by RNAseq from the livers of 20-21 week-old female C57BL/6J mice. Multiple, interacting factors were investigated: 1) To understand how euthanasia protocols affect gene expression when mouse carcasses are slow frozen, mice were euthanized by either euthasol injection, ketamine/xylazine injection, or CO2 inhalation, and carcasses slow frozen on dry-ice mimicking carcass preservation in the MELFI on the ISS. Carcasses were thawed and RNA extracted from livers; 2) To understand how carcass preservation protocols affect gene expression, mice were euthanized with euthasol and carcasses preserved by flash freezing in liquid nitrogen, slow freezing on dry ice, or immersion in RNAlater following three-way segmentation. Carcasses were thawed and RNA extracted from livers; 3) To understand how tissue preservation protocols affect gene expression, mice were euthanized with euthasol and livers dissected and processed immediately, or preserved by flash freezing in liquid nitrogen, slow freezing on dry ice, or immersion in RNAlater. Liver samples that were processed immediately were homogenized in RLT buffer and then either immediately further processed for RNA extraction or were stored for 70 days at -80C post-homogenization in sample RLT buffer prior to RNA extraction.