Single Cell RNA Amplification Nanodrop Processor

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DESCRIPTION (provided by applicant): Central nerve system (CNS) related diseases are one of the most prevalent causes of death and reduced life quality among human beings. In the effort to understand and combat CNS-related diseases, the ability to analyze single neural cells is an important tool distinct from global and regional analyses of the CNS in normal versus disease states, 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 utilizes digital electrodes to control discrete droplets that enable RNA isolation, cDNA amplification, biotin labeling, and target purification on a single chip. As the result, "microgram" of purified cDNA can be generated from "sub- picogram" of mRNA from a single cell. The RANP system enables automated processing on a very small volume for multi-step (20-25 steps) reactions, so that sample loading is the only manual step. During the Phase I project, the RANP-chip was designed, fabricated, and tested for cDNA synthesis, SPIA cDNA amplification, cDNA fragmentation, and biotin labeling. Approximately ~500,000-fold amplification was obtained from a few picogram of total RNA. The purified cDNA was in the range of 200-4000 nucleotides, with a peak around 500 bases and no detectable residual primers. This generated 248ng of purified amplified cDNA which is within our Phase I target. This technology offers the potential for robust identification of a single cell's transcriptional profile, which cannot be achieved with current technologies. Phase II work will focus on design and development of a prototype RANP chip, optimization of the nanodrop chip, integration of the RANP system for total automation, optimization of the RANP-based bioassay, and establish the RANP-chip and bioassay specifications.