Nanoscale Approaches Allow Highly Sensitive Quantification of Alternative Splice Sites in Plant Genes

Alternative splicing (AS) is a process that produces different mRNA variants from the same gene by using varying splice sites. This increases proteome variability to greatly expand the information content of genomes. AS in plants expands genome functions. It is also involved in regulation of developmental processes such as flowering and responses to environmental factors (temperature, light and pathogens) in plants.  Our project is based on an approach to detect and quantify AS events using Surface Enhanced Raman Scattering (SERS). SERS involves the use of gold nanoparticles to enhance the Raman scattering signals generated from biomolecules within nanometer distance of these signal-enhancing gold nanoparticles. Based on preliminary work using Raman spectroscopy and gold nanoparticles coded with non-fluorescent Raman probes, we have shown that mRNA targets extracted from cells, can not only be detected but also quantified up to femto-molar sensitivity. To establish this technology for plants we chose Arabidopsis as a model for AS, because global expression studies are very detailed for this plant and allow to determine AS for several genes. Quality control of SERS based quantification is performed by microarray hybridization, qRT-PCR and deep sequencing of mRNA. The goal of the project is to develop a protocol that allows precise quantification of mRNA species on a small scale or even single cell level. The study will provide insights into the role of AS in plant development and in various plant stress responses.