Automated Tag Library Generation for Sequencing-Based Gene Expression Profiling

DESCRIPTION (provided by applicant): Gene expression profiling, the use of technology to detect cellular transcripts in a high-throughput and massively parallel fashion, has started to move from the solely hybridization-based methods used in microarray te chnology to sequencing-based technologies for their increased sensitivities (10-100 times) and their ability to quantify virtually all messenger RNAs (mRNAs), including unknown transcripts, alternatively spliced, as well as microRNAs. While some systems ex ist to handle sample preparation for microarray applications of gene expression, the precursory target preparation steps for sequencing-based gene expression profiling are performed manually. The very tedious procedure requires one skilled full-time person 3 days to perform more than 50 steps, including mRNA isolation, cDNA synthesis and purification, cDNA cleavage and adapter ligation, as well as PCR amplification and target purification. As the procedure involves numerous pipetting steps and reagent trans fers, it presents an increased risk of contamination and a high likelihood of manual error accumulation, which in turn creates lower confidence data. Currently there is no system available to automate this tedious workload. Therefore, Maxwell Sensors Inc. (MSI) proposes to develop an Automated Integrated RNA Amplifier (AIRA) chip and system for generating digital cDNA tag libraries from biological samples for sequencing-based gene expression analysis. The proposed AIRA platform combines electrowetting-based nanodroplet manipulation and mRNA amplification chemistry using an integrated labon- a-chip system. The proposed AIRA harnesses the power of electrowetting-based digital fluidics that not only can automate all the sample preparation steps by dispensing, t ransporting, splitting, merging, and mixing of droplets, but also enables reactions to be performed in discrete nanoliter quantities on a single chip. Tremendous labor and time will be saved. The AIRA offers the potential for robust generation of millions of transcript fragments that can be identified by massively parallel sequencing at a fraction of the current cost. PUBLIC HEALTH RELEVANCE: Automated Integrated RNA Amplifier (AIRA) chip and system will be developed for processing RNA from biological sam ples in an automated, reliable, and cost-effective manner. The processed RNA can be directly applied to existing cutting-edge sequencing technologies to create a global RNA profile. The system will benefit researchers who are seeking better diagnosis and c ures for diverse complex diseases.