RNAseq Analysis of Rodent Spaceflight Experiments Is Confounded by Sample Collection Techniques.

San-Huei Lai Polo,Rick B Chen,Shayoni Ray,Yi-Chun Chen,Valery Boyko,Amanda M Saravia-Butler,Jonathan M Galazka,Homer Fogle,Sigrid S Reinsch,Sylvain V Costes,Oana Marcu,Kaushik Chakravarty,Marie T Dinh

doi:10.1016/j.isci.2020.101733

Abstract

SummaryTo understand the physiological changes that occur in response to spaceflight, mice are transported to the International Space Station (ISS) and housed for variable periods of time before euthanasia on-orbit or return to Earth. Sample collection under such difficult conditions introduces confounding factors that need to be identified and addressed. We found large changes in the transcriptome of mouse tissues dissected and preserved on-orbit compared with tissues from mice euthanized on-orbit, preserved, and dissected after return to Earth. Changes due to preservation method eclipsed those between flight and ground samples, making it difficult to identify spaceflight-specific changes. Follow-on experiments to interrogate the roles of euthanasia methods, tissue and carcass preservation protocols, and library preparation methods suggested that differences due to preservation protocols are exacerbated when coupled with polyA selection. This has important implications for the interpretation of existing datasets and the design of future experiments.

Highlights

Spaceflight places multiple stresses upon the human body including altered gravity fields and exposure to cosmic radiation, which lead to health risks for spacefaring humans (Institute of Medicine, 2008)
Follow-on experiments to interrogate the roles of euthanasia methods, tissue and carcass preservation protocols, and library preparation methods suggested that differences due to preservation protocols are exacerbated when coupled with polyA selection
Preservation Method Is the Primary Driver of Gene Expression Variance in RR-1 Liver Samples To assess gene expression differences in liver samples from the RR-1 NASA Validation mission (Cadena et al, 2019; Globus and Galazka, 2015; Ronca et al, 2019), RNA was extracted from livers dissected from spaceflight (FLT) and ground control (GC) animals either immediately after euthanasia or from frozen carcasses after partial thawing and sequenced following polyA selection (Figure S1)

Summary

Introduction

Spaceflight places multiple stresses upon the human body including altered gravity fields and exposure to cosmic radiation, which lead to health risks for spacefaring humans (Institute of Medicine, 2008). Despite success of the rodent model, sample collection under such difficult conditions introduces confounding factors that need to be identified and addressed. These are related to hardware limitations, small sample size, and severe restraints on astronaut crew availability. Animals can either be euthanized onboard the ISS or returned to Earth alive The former is experimentally challenging but preserves the sample during exposure to microgravity, whereas the latter exposes the animal to re-entry stresses, and sampling occurs only after a variable lag between landing and euthanasia—essentially sampling re-adaptation to Earth conditions in addition to the response to spaceflight. Inconsistent handling of samples necessitates a clear understanding of how dissection and preservation protocols affect downstream data generation

Methods

Results

Discussion

Conclusion