A matrix-adapted RNA extraction strategy for targeted RT-qPCR analysis of periodontal ligament fibroblasts cultured on human amniotic membrane scaffolds.

Introduction: Human amniotic membrane (HAM) is a promising scaffold for periodontal tissue engineering, supporting favorable cellular responses of human periodontal ligament fibroblasts (HPDLFs). However, molecular characterization of cells cultured on HAM is frequently hindered by extracellular matrix-derived contaminants, leading to poor RNA yield and compromised purity that limit downstream gene expression analyses. This study aims to develop and validate a matrix-adapted RNA extraction strategy to enable reliable molecular profiling of HPDLFs cultured on HAM scaffolds. Methods: HPDLFs were seeded onto de-epithelialized HAM, and cell viability was confirmed prior to RNA isolation. A systematic, multi-phase optimization of a phenol-based extraction protocol (GENEzol™ TriRNA Pure Kit) was undertaken to mitigate matrix-associated interference. RNA yield and purity were assessed spectrophotometrically, with functional validation performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: The optimized protocol significantly enhanced RNA integrity and yield compared to the conventional method. Purity indices improved (A260/230: 0.73 ± 0.20 to 2.15 ± 0.40; A260/280: 1.41 ± 0.27 to 2.03 ± 0.18), while RNA concentration increased markedly. The proportion of samples meeting stringent quality criteria improved from 0% to 50%. Notably, mean RNA purity parameters were not significantly different from standard monolayer cultures for the selected measures assessed (p > 0.05). Conclusions: The approach provides a practical framework for improving RNA recovery and targeted RT-qPCR validation in HAM-based culture systems which could set precedent for improving molecular investigations in periodontal tissue engineering and other scaffold-based models.