University of California, Irvine

The team proposes a unique instrument (the Single Cell Analyzer, SCA) for analysis of mRNA levels (at copy numbers of 5 to 2,000 molecules) in space and time within a living cell.  Current understanding of disease and developmental processes is largely based on biochemical or gene expression studies in the whole organ or tissue, coupled with phenotypic analysis of mutations in the context of the whole organism.  The SCA quantitatively queries gene expression at the single cell level, permitting examination of biological processes at much greater resolution.  The team has demonstrated that an active atomic force microscope (AFM) probe chemically modified with DNA primers can be inserted into a cell, without destruction, to selectively capture mRNA via an applied attractive dielectrophoretic force, and subsequently quantified.  Since mRNA levels can be assessed without affecting cell viability, the same cell can be monitored over time.  The proposed instrument will have applications in areas ranging from developmental and systems biology to personalized medicine, cancer diagnosis and stem cell research, with the potential to transform cell biology much as PCR has revolutionized molecular biology.  While transcript levels can be measured in fixed samples, no technology exists today that can dynamically quantify mRNA expression in individual living cells.