Testing the Stability of Plasma Protein and Whole Blood RNA in Archived Blood of Loggerhead Sea Turtles, Caretta caretta.

Abstract

Sample storage conditions can affect accuracy and reproducibility of biological measurements. Storing samples rapidly at the lowest available temperatures is considered ideal but is not always feasible when sampling in remote and logistically challenging field conditions, as is often the case with sea turtles. The objective of this study was to examine the stability of plasma proteins and quality of whole blood RNA from loggerhead sea turtle samples collected as part of an eighteen-year-long curated specimen collection. These biological variables are often used to assess sea turtle health; therefore, it is necessary to maintain the integrity of these components during storage. Protein electrophoresis was conducted on heparinized plasma from individual turtles collected in 2018 (n = 3), 2008 (n = 3), and 2001 (n = 3). Plasma was also pooled from four turtles sampled in 2018 and subjected to various storage temperatures. Whole blood was collected in blood collection tubes containing sodium heparin or PAXgene tubes with an RNA preservative. These were subjected to different storage treatments that can possibly occur during logistically difficult field sampling. Following various treatments, plasma proteins showed minor differences across collection years and no differences among storage treatments were observed, even when exposed to 38°C for three hours. RNA quality was assessed from whole blood using an RNA integrity number (RIN). RINs were poor from sodium heparin tubes that were frozen and from PAXgene tubes after an extended thaw. High-quality RNA was obtained from sodium heparin tubes that were never frozen and from PAXgene tubes with freezing delayed by up to 11 days. Overall, these results indicate that plasma proteins remain stable over time and when exposed to undesirable storage conditions, and RNA degrades rapidly in sea turtle blood after freezing and when not properly preserved. These aspects are important to consider when planning sampling protocols and logistics for optimal long-term sample preservation.