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World J Biol Chem. Sep 5, 2026; 17(3): 121018
Published online Sep 5, 2026. doi: 10.4331/wjbc.121018
Improved stabilisation of human pancreas biopsies: Comparative evaluation of RNA preservation methods
Rhys R Pook, Abbie Hearne, Rachel A Botting, Samuel J Tingle, Minna Honkanen-Scott, James A M Shaw, Simi Ali, William E Scott III
Rhys R Pook, Abbie Hearne, Rachel A Botting, Samuel J Tingle, Minna Honkanen-Scott, James A M Shaw, Simi Ali, William E Scott III, Department of Regenerative Medicine, Stem Cells, and Transplantation, Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne NE2 4HH, England, United Kingdom
Rhys R Pook, Abbie Hearne, Rachel A Botting, Samuel J Tingle, James A M Shaw, Simi Ali, William E Scott III, NIHR Blood and Transplant Research Unit - Newcastle and Cambridge University, Newcastle University, Newcastle upon Tyne NE2 4HH, England, United Kingdom
Samuel J Tingle, James A M Shaw, Institute of Transplantation, Freeman Hospital, Newcastle upon Tyne NE7 7DN, England, United Kingdom
Author contributions: Pook RR drafted the original manuscript with each author editing and contributing during revisions, and designed the study and formulated the research idea; Pook RR, Hearne A, and Honkanen-Scott M performed the organ biopsies and processed the samples; Pook RR, Hearne A, and Botting RA acquired, analysed and interpreted the data; Tingle SJ contributed to statistical analysis and R code development; Scott III WE, Ali S, and Shaw JAM supervised or mentored the study; and all authors thoroughly reviewed and endorsed the final manuscript.
AI contribution statement: ChatGPT (OpenAI) was used to assist with debugging and troubleshooting R code used in this study. AI tools were not used to generate research data, interpret results, or draw conclusions. All AI-assisted content was critically reviewed and edited by the authors.
Supported by the National Institute for Health and Care Research Blood and Transplant Research Unit in Organ Donation and Transplantation, No. NIHR203332.
Institutional review board statement: This study was approved by the Medical Ethics Committee of NHS Blood and Transplant North East - Newcastle and North Tyneside 1 Research, approval No. 16/NE/0230.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: Any data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Corresponding author: William E Scott III, PhD, Associate Professor, Department of Regene
rative Medicine, Stem Cells, and Transplantation, Translational and Clinical Research Institute, Newcastle University, E1.08 Biomedicine West Wing, The International Centre for Life, Times Square, Scotswood Road, Newcastle upon Tyne NE2 4HH, England, United Kingdom.
bill.scott3@newcastle.ac.uk
Received: March 23, 2026
Revised: May 13, 2026
Accepted: June 23, 2026
Published online: September 5, 2026
Processing time: 166 Days and 14.5 Hours
BACKGROUND
The pancreas is particularly vulnerable to rapid post-retrieval degradation due to its high endogenous RNase and enzymatic activity. This presents a major challenge in both pancreas research and clinical transplantation for accurate transcriptomic analyses. Accurate molecular profiling is increasingly important for evaluating graft quality and characterising injury. Although RNA preservation strategies have been reported in animal tissue studies, their comparative performance in human pancreatic tissue remains under-reported.
AIM
To evaluate biopsy preservation methods to validate an optimal approach capable of stabilising human-pancreas biopsies for high-quality gene expression analysis.
METHODS
Four clinically declined human pancreata were regionally sampled (36 biopsies per pancreas). Tissue was preserved using either: (1) Immediate RNA isolation; (2) Snap freezing; (3) RNAlater submersion; or (4) RNAlater injection. Immediate samples were extracted the same day with snap frozen and RNAlater samples being snap frozen and freeze-thawed prior to extraction using a spin-column protocol. RNA concentration and purity were assessed by Nanodrop and RNA integrity number (RIN) generated using a TapeStation. Statistical analyses were conducted using R-Studio.
RESULTS
All samples bar one yielded RNA of acceptable concentration (25-500 ng/μL) and purity (260/280 ~2.00, 260/230 2.00-2.2). RIN values varied significantly. Both RNAlater injection (7.1 ± 1.10; adjusted P value = 0.0089) and RNAlater submersion (7.1 ± 1.13; adjusted P value = 0.0058) produced significantly higher RIN scores compared to snap freezing (3.7 ± 1.21). RNAlater preserved samples exceeded the minimum RIN threshold for downstream transcriptomic analysis (RIN ≥ 7.0). No significant difference was observed between RNAlater techniques or immediate isolation (5.0 ± 1.18).
CONCLUSION
RNAlater based preservation by submersion or injection provided superior stabilisation of human pancreatic RNA compared with conventional snap freezing. RNAlater preserves the transcriptome at the moment of tissue acquisition, minimising degradation post biopsy. Despite the wide adoption of RNAlater usage, this study provides confirmation that RNAlater reliably supports high-quality RNA extraction from biopsies filling the current gap in pancreas-specific evidence. Implementation of this method may enable more accurate evaluation of graft injury, improve biomarker development, and support high-quality biobanking of human pancreas biopsies for future studies.
Core Tip: Obtaining high-quality RNA from human pancreatic tissue remains a persistent challenge in transplant research due to its high endogenous RNase activity and unavoidable pre-analytical ischemia. Using 144 biopsies from four deceased donor pancreata, we systematically evaluated tissue preservation strategies. Our findings demonstrate that both tissue submersion and direct injection of RNAlater yield significantly superior RNA integrity compared with conventional methods. This study provides specific human evidence for a simple and scalable strategy to ensure reliable biobanking and downstream molecular analysis of human pancreatic tissue.
