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

Abstract

To 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.

Acute stress induces severe neural inflammation and overactivation of glucocorticoid signaling in interleukin-18-deficient mice

“…4A . Seventeen genes with a false discovery rate <0.30 were extracted from the RNA-Seq results based on previous study [ 32 , 33 ], and nine genes were assigned GenBank accession numbers (Table 1 , Supplementary Table 2 ). RT-qPCR was performed to confirm the RNA-Seq results (Fig.…”

NASA GeneLab RNA-seq consensus pipeline: Standardized processing of short-read RNA-seq data

“…In addition, the pipeline cannot compensate for poor library preparation technique or inadequate sample size. Sample preservation protocols between datasets need to also be evaluated, because variations in sample preservation protocol could lead to poor correlation between studies that are otherwise identical ( Lai Polo et al, 2020 ). The number of sequenced reads may also be a limiting factor in the usefulness and accuracy of the differentially expressed genes calculated by DESeq2 and, similarly, during splice isoform analysis.…”

Challenges and considerations for single-cell and spatially resolved transcriptomics sample collection during spaceflight

“… 69 Another study compared bulk RNA-seq profiles between mouse liver tissue dissected and frozen directly on the ISS compared to mice preserved as frozen whole carcasses with their liver tissue dissected back on Earth. 70 This study notes that whole-carcass preservation in RNAlater can better preserve the tissues; however, there are a few caveats to this approach. The first caveat is that RNAlater has only had limited success in preserving cells for scRNA-seq on Earth 71 and will require further optimization before its application can be recommended for such experiments in spaceflight.…”