There has been an important realization over the past several years that in order to truly understand human disease we must understand not just the diseased tissues, but rather the cells in those tissues. To that end, we need tools that can capture those cells and explore their expression profiles. We have developed a technology called xMD-miRNA-Seq to do just that. The xMD stands for expression microdissection and it allows us to collect one cell type at a time from any fixed tissue source. We have developed a way to use those cells for small RNA sequencing to capture the expression profile of microRNAs from just these cells. microRNAs (miRNAs) are small, regulatory RNAs involved in disease and development. Although this is an exciting, powerful technique designed to help understand cell expression, it remains relatively inefficient. Therefore, we have proposed a set of optimizations to make the technique widely adaptable. We have proposed to optimize the capture of cells moving from a slide to an affixed membrane by adjusting features including the temperature, the light intensity, the background type and even the membrane itself. We also seek to limit the loss of RNA through the steps of the xMD process by pretreating certain reagents with chemicals designed to remove RNAses. Finally, we will work with both established and new methods to maximize the percent of miRNAs that are read in a small RNA sequencing run. Ultimately, these modifications will greatly enhance the value of the xMD-miRNA-seq method and will allow us to obtain the miRNA profile of almost any human cell type in almost any disease setting. This will be important for cancer biology, immunology, development, cardiovascular disease, neurologic disorders and many other settings.
This project will develop and optimize a new technology to directly determine the expression of microRNAs from cells still within tissue. Multiple methodologic advances will be made to turn our method into a cost- effective, robust and easy to run technology. This technology can be used to study a wide range of human diseases in an entirely new way.
