Techniques for the isolation of high-quality RNA from cells encapsulated in chitosan hydrogels

Extracting high-quality RNA from hydrogels containing polysaccharide components is challenging, as traditional RNA isolation techniques designed for cells and tissues can have limited yields and purity due to physiochemical interactions between the nucleic acids and the biomaterials. In this study, a comparative analysis of several different RNA isolation methods was performed on human adipose-derived stem cells photo-encapsulated within methacrylated glycol chitosan hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy® mini kit resulted in low yields of RNA, except when the samples were preminced directly within the buffer. In addition, genomic DNA contamination during reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was observed in the hydrogels processed with the CTAB-based methods. Isolation methods using TRIzol® in combination with one of a Qiaex® gel extraction kit, an RNeasy® mini kit, or an extended solvent purification method extracted RNA suitable for gene amplification, with no evidence of genomic contamination. The latter two methods yielded the best results in terms of yield and amplification efficiency. Predigestion of the scaffolds with lysozyme was investigated as a possible means of enhancing RNA extraction from the polysaccharide gels, with no improvements observed in terms of the purity, yield, or amplification efficiency. Overall, this work highlights the application of a TRIzol®+extended solvent purification method for optimizing RNA extraction that can be applied to obtain reliable and accurate gene expression data in studies investigating cells seeded in chitosan-based scaffolds.