Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.

提供生物学相关微环境的三维 (3D) 细胞培养模型对于体外研究细胞-细胞和细胞-基质相互作用至关重要. 半合成星形聚（乙二醇）（starPEG）-肝素水凝胶因其高度可调的生化和生物力学特性而被广泛用于 3D 细胞培养。基因表达水平的变化通常用作衡量细胞反应的指标。然而，高质量 RNA 的分离带来了挑战，因为 RNA 被水凝胶残留物（如聚合物或糖胺聚糖片段）污染会影响模板的质量和数量，从而限制有效的基因表达分析。在这里，我们比较了两种从 starPEG-肝素水凝胶中提取高质量 RNA 的方案，并评估了三种后续的纯化技术。发现通过离心去除水凝胶残留物对于在两种分离方法中获得高质量 RNA 是必不可少的。然而，RNA 的纯化并没有导致 RNA 质量的进一步提高。此外，我们展示了提取的 RNA 对通过定量聚合酶链反应 (qPCR) 确认的三个内源性对照基因的 cDNA 合成的适用性。所示的方法和技术可以针对基于天然或半合成材料的其他水凝胶模型进行定制，从而为所有基因表达分析提供强大的模板。