PH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

Abstract

Chitosan has emerged as a useful biomaterial employed in tissue engineering and drug delivery applications due to its tunable and interesting properties. However, chitosan is protonated at biological pH and thus carries positive charges, which renders chitosan incompatible with conventional methods of RNA extraction. RNA extraction is an important step in investigating cell responses and behavior through studying their gene expression transcriptional profiles. While some researchers have tried different techniques to improve the yield and purity of RNA extracted from cells encapsulated in chitosan-based biomaterials, no single study has investigated the effects of manipulating pH of the homogenate during RNA extraction on the yield and quality of total RNA. This study confirms the release and binding of RNA from chitosan to be pH dependent while analyzing the impact of pH changes during the tissue disruption and homogenization step of extraction on the resulting yield and quality of isolated RNA. This concept was applied to three commonly used methods of RNA extraction, using adult neural stem/progenitor cells (aNSPCs) encapsulated within methacrylamide chitosan (MAC) as a model chitosan-based bioscaffold. High pH conditions resulted in high yields with good quality using both TRIzol and CTAB. pH of the homogenate did not affect RNeasy spin columns, which worked best in neutral conditions with good quality, however, the overall yield was low. Results in total show that pH affected RNA interaction with a chitosan-based bioscaffold, and thus altered the concentration, purity, and integrity of isolated RNA, dependent on the method used.