Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments

Abstract

BackgroundEven with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved.ObjectivesThe goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C.MethodsTissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH).ResultsFresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6–7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months.ConclusionThese results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.

Upregulation of genes encoding digestive enzymes and nutrient transporters in the digestive system of broiler chickens by dietary supplementation of fiber and inclusion of coarse particle size corn

“…An RNA 6000 Nano kit was used to measure RNA integrity (RNA Integrity Number, or RIN) using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany). The RNA samples were considered of high integrity if the RIN was higher than 7.5 [ 21 ]. The RIN values of the samples were 7.9–9.8 in the present study.…”

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

“…With the rise of RNAlater (Ambion, Invitrogen) as a popular storage method in field‐based studies (De Smet et al, ; Wille et al, ), it is important to quantify if there are systematic biases in gene expression when samples are preserved in RNAlater vs. flash‐frozen in liquid nitrogen. In our literature review, however, we could find few direct comparisons of RNAseq data obtained from the most common field preservation method RNAlater and the “gold standard” of flash freezing samples in liquid nitrogen (Alvarez, Schrey, & Richards, ; Wolf, ; but see Cheviron, Carling, & Brumfield, ; Choi, Ray, Lai, Alwood, & Globus, ). Further, few studies examine whether a systematic bias due to gene characteristics exists for samples preserved in RNAlater (Bray et al, ).…”

RNAseq analysis of rodent spaceflight experiments is confounded by sample collection techniques

“…Since livers from only 2 FLT and 2 GC animals in the RR-1 NASA Validation mission were preserved via the immediate method, RNA from livers prepared via the immediate method from two additional studies, the RR-1 CASIS mission (Cadena et al, 2019;Ronca et al, 2019) and a ground-based preservation and storage study (Choi et al, 2016;GeneLab, 2016) were also sequenced following polyA selection. Despite multiple different experimental factors in RR-1 NASA, RR-1 CASIS, and the ground-based preservation studies, PCA continued to show preservation method as the primary driver of variance among samples in these datasets ( Figure S1).…”