Central nerve system (CNS) related diseases are one of the most prevalent causatives of death and reduced life quality among human beings. In an effort to understand and combat CNS-related diseases, the ability to analyze single neural cells is an important distinction from global and regional analyses of the CNS in the state of normal function versus disease, as each neural cell has a unique molecular signature. However, single-cell gene expression profiling is currently hampered due to the low amount of messenger RNA (mRNA) present in a single cell. The quantity of mRNA harvested from a single cell, estimated to be approximately 0.1-0.2 picograms, is below the level of sensitivity for standard RNA extraction procedures and is likely to have losses during the preamplification processes. Furthermore, because of the dilution of the mRNA templates and reduction in enzymatic efficiency, it often results in biased data, which affects biological interpretation. ? ? Therefore, Maxwell Sensors Inc. proposes to develop an RNA Amplification Nanodrop Processor (RANP) for parallel processing of global mRNA from single mammalian cells. The proposed RANP platform combines three technologies: nanodroplet manipulation, electro-wetting, and mRNA extraction/amplification chemistry into an integrated lab-on-a-chip system. Nanodrop manipulation enables reactions to be performed in a discrete nanoliter quantity, while electro-wetting offers the direct liquid manipulation, including dispensing, transporting, splitting, merging, and mixing of droplets. In combination of these technologies, the RANP system will automate cell lysis, RNA isolation, DNA amplification, biotin labeling, and target purification from a single cell. The resulting system will minimize loss of mRNA template through automation of all steps of RNA processing, minimize stochastic effects by controlled manipulation of nanoliter volumes, process hundreds of single cells simultaneously on a single chip, and increase the consistency and reliability of downstream microarray assays for single-cell profiling. It offers the potential to robustly identify a single cell's transcriptional profile, which is not able to be achieved with current technologies. ? ? ?
