Improved stabilisation of human pancreas biopsies: Comparative evaluation of RNA preservation methods

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.

To evaluate biopsy preservation methods to validate an optimal approach capable of stabilising human-pancreas biopsies for high-quality gene expression analysis.

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.

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).

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.