Optimization of RNA Isolation Methodology from Three-Dimensional Collagen Hydrogel Culture Systems
Poster Number
105
Session Title
Biology and Biomedical Research
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Matthew Stern, Ph.D.
Abstract
Three-dimensional culture systems allow for more complex cellular interactions and organization than traditional two-dimensional culture, which better replicates an in vivo environment. Interestingly, cells placed on top of collagen hydrogels organize into a donut-like shape called a toroid, while cells mixed into the hydrogels do not organize into higher order structures. The goal of our project is to evaluate the signal transduction pathways and cellular mechanisms that mediate toroid formation. The specific goal of the work described here was to optimize RNA isolation methodology from cells cultured on or in collagen hydrogels – a procedure that is known to be technically challenging – and to use real-time RT-PCR to compare the expression of select genes during toroid formation. We tested and compared several different RNA isolation protocols and found that a method based on the use of cetyltrimethylammonium bromide (CTAB) prior to alcohol precipitation, which is more typically used in isolation of RNA from plants, proved to be the most consistently effective in our hands. We went on to conduct a twelve-hour time-course experiment where RNA was isolated from adipose derived stem cells cultured under toroid-forming conditions every two hours. This enables us to compare gene-expression profiles of cells during toroid formation via methods like real-time RT-PCR andRNA sequencing in the future. Such comparisons will provide valuable insight into the mechanisms of toroid formation and self-organization by stem cells in developmental and regenerative contexts.
Grant Support?
Supported by a grant from the South Carolina EPSCoR/IDeA Collaborative Research Program and by an SC INBRE grant from the National Institute for General Medical Sciences (NIH-NIGMS)
Start Date
24-4-2020 12:00 AM
Optimization of RNA Isolation Methodology from Three-Dimensional Collagen Hydrogel Culture Systems
Three-dimensional culture systems allow for more complex cellular interactions and organization than traditional two-dimensional culture, which better replicates an in vivo environment. Interestingly, cells placed on top of collagen hydrogels organize into a donut-like shape called a toroid, while cells mixed into the hydrogels do not organize into higher order structures. The goal of our project is to evaluate the signal transduction pathways and cellular mechanisms that mediate toroid formation. The specific goal of the work described here was to optimize RNA isolation methodology from cells cultured on or in collagen hydrogels – a procedure that is known to be technically challenging – and to use real-time RT-PCR to compare the expression of select genes during toroid formation. We tested and compared several different RNA isolation protocols and found that a method based on the use of cetyltrimethylammonium bromide (CTAB) prior to alcohol precipitation, which is more typically used in isolation of RNA from plants, proved to be the most consistently effective in our hands. We went on to conduct a twelve-hour time-course experiment where RNA was isolated from adipose derived stem cells cultured under toroid-forming conditions every two hours. This enables us to compare gene-expression profiles of cells during toroid formation via methods like real-time RT-PCR andRNA sequencing in the future. Such comparisons will provide valuable insight into the mechanisms of toroid formation and self-organization by stem cells in developmental and regenerative contexts.
