Automated Tag Library Generation for Sequencing-Based Gene Expression Profiling

描述（由申请人提供）：基因表达谱，利用技术以高通量和大规模并行的方式检测细胞转录物，已经开始从微阵列技术中使用的完全基于杂交的方法转向基于测序的技术，因为它们的灵敏度增加（10-100倍），并且它们能够量化几乎所有的信使rna (mrna)，包括未知转录物，选择性剪接，以及microRNAs。虽然存在一些系统来处理基因表达微阵列应用的样品制备，但基于测序的基因表达谱的前期目标制备步骤是手动执行的。这是一个非常繁琐的程序，需要一个熟练的全职人员3天完成50多个步骤，包括mRNA分离，cDNA合成和纯化，cDNA切割和适配器连接，以及PCR扩增和目标纯化。由于该过程涉及许多移液步骤和试剂转移，因此污染风险增加，人工错误积累的可能性很高，这反过来又会产生较低的置信度数据。目前还没有系统可以自动完成这项繁琐的工作。因此，Maxwell Sensors Inc. （MSI）提出开发一种自动集成RNA放大器（AIRA）芯片和系统，用于从生物样品中生成数字cDNA标记文库，用于基于测序的基因表达分析。提出的AIRA平台结合了基于电润湿的纳米液滴操作和使用集成labon- a芯片系统的mRNA扩增化学。提出的AIRA利用基于电润湿的数字流体的力量，不仅可以通过分配，运输，分裂，合并和混合液滴来自动化所有样品制备步骤，而且还可以在单个芯片上以离散的纳升数量进行反应。将节省大量的劳动和时间。AIRA提供了生成数百万个转录本片段的潜力，这些转录本片段可以通过大规模平行测序来识别，而成本仅为当前成本的一小部分。公共卫生相关性：将开发自动集成RNA放大器（AIRA）芯片和系统，以自动化、可靠和经济有效的方式处理生物样品中的RNA。经过处理的RNA可以直接应用于现有的尖端测序技术，以创建全球RNA图谱。该系统将使那些为各种复杂疾病寻求更好的诊断和治疗方法的研究人员受益。

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 technology 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 exist 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 transfers, 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, transporting, 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 samples 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 cures for diverse complex diseases.