• Systematic comparison of RNA extraction methods for hydrogel-encapsulated organoids • Alginate–norbornene hydrogels interfere with RNA yield, purity, and qPCR performance. • Alginate lyase digestion improves RNA extraction consistency from encapsulated organoids. • Plant RNA magnetic bead-based kit enables robust gene expression analysis after hydrogel removal. • Optimized workflow supports reliable RNA isolation from alginate-norbornene encapsulated organoids. The encapsulation of kidney organoids within hydrogels provides a biomimetic environment that enhances their structural and functional relevance for disease modeling and drug screening. However, the presence of hydrogel matrices poses a major challenge for molecular analysis, particularly for RNA extraction, where residual material can interfere with yield, purity, and downstream applications. The objective of this study was to systematically evaluate RNA extraction methods for kidney organoids encapsulated in alginate-norbornene hydrogels and identify an optimized protocol suitable for reliable gene expression analysis. We compared commonly used extraction methods designed for mammalian tissues and plant-derived materials, with and without prior enzymatic digestion of the hydrogel. RNA yield and purity were assessed by spectrophotometry and fluorometry, while RNA integrity was analyzed by Bioanalyzer, and performance in downstream assays was evaluated by quantitative PCR of housekeeping genes. Our results showed that RNA yield was consistently lower in encapsulated organoids compared to suspension cultures, reflecting smaller organoid size and reduced metabolic activity in encapsulated conditions. Spectrophotometric purity ratios differed between suspension and encapsulated samples, but RNA integrity was preserved across all methods, with values within the acceptable range. Quantitative PCR revealed that TRIzol-based extractions introduced significant variability between suspension and encapsulated samples. Conversely, the protocol with alginate lyase digestion followed by the Maxwell RSC RNA kit produced the most reproducible results. Ct values for control and encapsulated samples were highly consistent, with inter-condition variability remaining below 0.5 standard deviations across replicates. These findings highlight the importance of adapting RNA isolation protocols to account for the presence of hydrogels. Alginate lyase digestion combined with a plant RNA extraction kit offers a reliable strategy for obtaining high-quality RNA from encapsulated kidney organoids within alginate-based hydrogels. While this approach enabled a robust gene expression analysis providing a foundation for transcriptomic studies, different hydrogels and organoid combinations might require additional adjustments underscoring the importance of adopting methods to ensure optimal RNA quality for downstream methods.
Kearney et al. (Sun,) studied this question.