Temporal Postmortem RNA Degradation in Subcutaneous Adipose Tissue in Swine

Abstract Text: When collecting tissues for RNA analysis, it is generally accepted that samples should be preserved immediately postmortem to limit RNA degradation. However, this often is challenging in practical situations. While RNA degradation in many tissues has been previously investigated, RNA degradation in postmortem adipose tissue is less understood. The objective of this study was to characterize qualitative and quantitative changes in RNA from adipose tissue with increasing time postmortem. Subcutaneous adipose samples were obtained from five pigs at nine time points, ranging from immediately after exsanguination to six days postmortem. Samples were flash frozen in liquid nitrogen, then stored at -80ºC until RNA extraction. An Agilent Bioanalyzer and Nanodrop spectrophotometer were used to determine RNA quality. Expression of key adipogenic genes including stearoyl-coA desaturase (SCD), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin (ADIPOQ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by qPCR. Data were analyzed as repeated measures using the mixed procedure of SAS. The 260/280 ratio was unaffected (P = 0.41) by time postmortem. RNA integrity number (RIN) decreased (P < 0.01) over time, with samples collected at 2 hours postmortem similar (P > 0.05) to samples collected immediately after exsanguination. Additionally, cycles to threshold (CT) increased (P > 0.01) for all genes over time, an indication of increased RNA degradation over time. However, for each gene, CT of samples collected at 0 min, 30 min, and 2 hours after exsanguination were not different (P > 0.05). When PPARγ was normalized to GAPDH, ΔCT values were unaffected by time (P > 0.05). However, normalized to GAPDH, both SCD and ADIPOQ ΔCT values increased (P < 0.01) over time, indicating that SCD and ADIPOQ degrade at a faster rate than GAPDH. For SCD, ΔCT increased (P < 0.01) at 24 hours compared to the sample taken immediately after exsanguination while samples taken before this time were all similar (P > 0.05).  For ADIPOQ, ΔCT was increased (P < 0.05) at 5 hours compared to the initial time point, while samples taken before 5 hours were similar (P > 0.05) to the initial sample. Therefore, while subcutaneous adipose samples collected within 2 hours postmortem are optimal, samples could be collected up to 5 hours postmortem and avoid biases in RNA degradation with respect to genes evaluated in this study. 

Keywords: adipose, RNA, swine, postmortem degradation, SCD, PPARγ, ADIPOQ, GAPDH