Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Optimization of RNA Isolation Methodology for Gene Expression Analysis of Self-Organizing Three-Dimensional Tissue Structures

Three-dimensional culture systems allow for more complex cellular interactions and organization than traditional two-dimensional culture. It has been observed that 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 toroids. The ultimate goal of this project is to evaluate the signal transduction pathways and cellular mechanisms that are involved in toroid formation. The specific goal of the work described here was to optimize RNA isolation 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. These results allowed us to move onto using real-time RT-PCR to compare the expression of two specific genes, Cxcr4 and Cxcl12 in mouse adipose-derived stem cells over a 12-hour timecourse of toroid formation. Our results revealed some differences in gene expression during toroid formation. More importantly, they demonstrate the feasibility of conducting similar and/or larger-scale experiments using methods like RNA-sequencing to monitor changes in gene expression during toroid formation and to compare gene expression between different collagen hydrogel-based culture platforms.