Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

Abstract

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 photoencapsulated within methacrylated glycol chitosan hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy Ò 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 Ò in combination with one of a Qiaex Ò gel extraction kit, an RNeasy Ò 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 Ò + 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.

Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization

“…Our results are similar to the data reported by Yu et al on RNA isolation from cells encapsulated in chitosan hydrogels, confirming that the use of an enzymatic pretreatment improves the purity of samples isolated with guanidinium thiocyanate . In contrast to this previous work, enzymatic pretreatment with pronase resulted in good yield, and high 260/280 and 260/230 absorption ratios without the need of further purification using RNA columns.…”

“…Ideally, the chosen RNA isolation method should allow the isolation of high‐quality, intact RNA from all tissue types. Several methods have been reported for the isolation of RNA from collagen‐rich tissues such as cartilage, and polysaccharide‐rich materials such as plants and chitosan‐based hydrogels . The main challenge of isolating RNA from IVD (and NP in particular) is the combination of low cellularity and high proteoglycan content.…”

“…There have been other techniques described for the isolation of RNA from disc tissue which can avoid the problem of proteoglycan coprecipitation, such as enzymatic tissue digestion, although the yields from single discs are relatively low. Additionally, specific protocols have been developed for the isolation of RNA from cells cultured in polysaccharide‐based hydrogels . However, these techniques may not be as successful when applied to disc tissue, which has an even higher proteoglycan content and lower cellularity.…”

Peptide‐modified methacrylated glycol chitosan hydrogels as a cell‐viability supporting pro‐angiogenic cell delivery platform for human adipose‐derived stem/stromal cells

“…ASCs cultured in 2‐D on TCPS were included as a calibrator. Total RNA was extracted following established methods using TRIzol reagent and the RNeasy® kit (Qiagen, Toronto, ON) . Post‐extraction RNA clean‐up was performed by adding 0.1× the volume of 3 M sodium acetate, 2.5× the volume of 100% ethanol, and 0.01× the volume of 5 mg/mL glycerol to the aqueous RNA sample.…”

Chondrocyte Generation of Cartilage‐Like Tissue Following Photoencapsulation in Methacrylated Polysaccharide Solution Blends

“…DNA content was measured using the Hoechst 33258 dye‐binding assay . However, due to binding of DNA to the hydrogel material, the DNA was approximated by using the (3‐(4,5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide (MTT) assay . Following previously reported protocols a standard curve was used to convert the MTT absorbance values for each hydrogel into a cell number and then multiplied by 5.6 ± 0.1 pg DNA/chondrocyte to determine the total DNA accumulated within the hydrogels .…”