Factors That Influence the Quality of RNA From the Pancreas of Organ Donors

Abstract

Objectives Attaining high quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, Methods RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and utilized to define high (≥6.5) and low (≤4.5) quality RNA. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. Results Univariate analysis revealed donor cause of death (Odds Ratio [OR]=0.35, 95% Confidence Interval [CI]=0.15–0.77, p=0.01), prolonged tissue storage prior to RNA extraction (OR=0.65, 95%CI 0.52–0.79, p<0.01), pancreas region sampled (multiple comparisons, p<0.01), and sample type (OR=0.32, 95%CI 0.15–0.67, p<0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR=3.95, 95%CI 1.59–10.37, p<0.01) and sample collection protocol (OR=8.48, 95%CI 3.96–19.30, p<0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared to total pancreatic RNA from the same donor (∆RIN=1.3, 95%CI 0.6–2.0, p<0.01). Conclusions A multivariable model demonstrates that autopsy- and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA.

Enteroviral Infections as a Trigger for Type 1 Diabetes

“…Partial EV sequences were identified but no specific genotype could be determined [7••]. This illustrates how challenging the detection of low abundance RNA and protein in the pancreas can be and how the use of optimized and standardize tissue collection and processing methods is key toobtain high-quality data[26].…”

Organ donor pancreases for the study of human islet cell histology and pathophysiology: a precious and valuable resource

“…Although the process from donor hospital admission to pancreas retrieval cannot always be fully controlled, the duration of hospital stay until brain death (a major step in the organ donation timeline), does not seem to be associated with significant changes in key histological features of the retrieved pancreas, such as leucocyte and macrophage infiltration, and frequency of replicating beta cells. In addition, in a previous study, duration of final hospitalisation was shown not to be negatively associated with the quality of nucleic acid extracted from organ donor pancreases [38]. However, it must be noted that all the studies that have investigated the correlation between hospital time before brain death and pancreatic histopathological features have been conducted using tissue from donors without diabetes and it is not known whether similarly encouraging findings are applicable to organ donors with diabetes.…”

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

“…Benchmarking, quality assurance and control standards, and standard operating procedures, where appropriate, will be developed for each stage of the methodological process and be made available to promote rigor, reproducibility and transparency. It is expected that quality assurance and control standards for both biospecimens and data will evolve as tissue collection, processing techniques, storage and shipping conditions, assays, and data-processing tools change, and as HuBMAP interacts and collaborates with other related efforts, as they have for other consortium projects 26–31 . Where possible, metadata related to preanalytical variables (for example, annotations and nomenclature) and technologies will be harmonized, and protocols and standards will be shared with the wider research community.…”