Optimization of a Method of Nucleic Acid Extraction from Three-Dimensional Hydrogel Cultures
Session Title
Biology and Genetics
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Matthew Stern, Ph.D.
Abstract
The ability to provide a 3D structure for cellular growth is important for tissue engineering. Previous studies conducted by our collaborators have found that essentially any type of non-cancerous cell will form a toroidal structure within 24 hours of being placed on top of a collagen hydrogel. However, when the cells are mixed into the collagen hydrogel prior to geleation, they remain as a more diffuse network of cells 24 hours later. In contrast, all types of cancerous cells tested fail to form toroids when plated on top of a collagen hydrogel. We are interested in identifying the molecular mechanisms that contribute to the toroidal organization of normal cells and which component(s) are lacking and/or misregulated in cancer cells. One major challenge the project has faced is the inability to obtain sufficient amounts of nucleic acids (both RNA and DNA) from the toroids and gels. Our goal for this part of the project was to optimize methodology for extracting RNA from the different experimental set-ups. High quality RNA is required for real time PCR analyses of genes suspected to be involved in the organization of cells and for prospective identification of differences in gene expression between normal and cancerous cells via RNA sequencing and/or microarray. We show that a hybridized method of RNA extraction yields workable quantities of high purity RNA that can be used in the desired downstream applications.
Grant Support?
Supported by a grant from the National Science Foundation and the South Carolina EPSCoR/IDeA Program
Start Date
12-4-2019 3:30 PM
Optimization of a Method of Nucleic Acid Extraction from Three-Dimensional Hydrogel Cultures
WEST 219
The ability to provide a 3D structure for cellular growth is important for tissue engineering. Previous studies conducted by our collaborators have found that essentially any type of non-cancerous cell will form a toroidal structure within 24 hours of being placed on top of a collagen hydrogel. However, when the cells are mixed into the collagen hydrogel prior to geleation, they remain as a more diffuse network of cells 24 hours later. In contrast, all types of cancerous cells tested fail to form toroids when plated on top of a collagen hydrogel. We are interested in identifying the molecular mechanisms that contribute to the toroidal organization of normal cells and which component(s) are lacking and/or misregulated in cancer cells. One major challenge the project has faced is the inability to obtain sufficient amounts of nucleic acids (both RNA and DNA) from the toroids and gels. Our goal for this part of the project was to optimize methodology for extracting RNA from the different experimental set-ups. High quality RNA is required for real time PCR analyses of genes suspected to be involved in the organization of cells and for prospective identification of differences in gene expression between normal and cancerous cells via RNA sequencing and/or microarray. We show that a hybridized method of RNA extraction yields workable quantities of high purity RNA that can be used in the desired downstream applications.
