Optimization of a Protocol for Escherichia coli RNA Extraction and Visualization

We attempted to improve a protocol to allow for the consistent extraction of total RNA from prokaryotic cells. Total RNA was isolated from Escherichia coli B23 and yeast cells using Trizol. In the comparison of two different protocols it was found that performing the phase separation independent from the bead bashing results in increased yield and purity of the RNA. We also attempted to improve resolution of the low molecular weight RNA molecules by comparing samples of RNA run on agarose and polyacrylamide gels. The polyacrylamide gels proved superior in resolving 5S, 5.8S rRNA and tRNA. Degassing significantly improved the well formation, resulting in more distinct bands. A comparison of glyoxal and formaldehyde denaturants was inconclusive, but preliminary findings suggest that formaldehyde has better potential. The use of yeast as an internal standard for prokaryotic RNA extraction was unsuccessful, but shows promise for future investigation.