Rapid mRNA Expression Analysis by Quantitative Electrochemical Microarray at Sub-Zeptomole Levels without PCR and Labels

通过定量电化学微阵列在亚 Zeptomole 水平上进行快速 mRNA 表达分析，无需 PCR 和标记

• 批准号: 9556918
• 负责人: Ravi Saraf
• 金额: $ 28万
• 依托单位: VAJRA INSTRUMENTS, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9556918
• 关键词: Address; Binding; Biological Process; Biology; Body Fluids; Buffers; Calibration; Cell Line; Cell physiology; Cells; Complementary DNA; Data; Data Quality; Detection; Diagnostic; Disease; Dyes; Electrochemistry; Electrodes; Expression Profiling; Fine needle aspiration biopsy; Fluorescence; Gene Expression; Gene Mutation; Genes; Genetic; Genomics; Goals; Gold; Healthcare; Kinetics; Label; Lasers; Measurement; Measures; Mediating; Medicine; Messenger RNA; Methods; Methylene blue; Microarray Analysis; Molecular; Natural regeneration; Nature; Noise; Nucleic Acids; Nucleotides; Outcome; Oxidation-Reduction; Pattern; Performance; Phase; Process; RNA; RNA-Directed DNA Polymerase; Reaction; Reading; Research; Scanning; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Speed; Spottings; Technology; Therapeutic; Time; Variant; base; gene discovery; interest; invention; logarithm; mRNA Expression; prognostic; statistics; stem; success; transcriptome; Address; Binding; Biological Process; Biology; Body Fluids; Buffers; Calibration; Cell Line; Cell physiology; Cells; Complementary DNA; Data; Data Quality; Detection; Diagnostic; Disease; Dyes; Electrochemistry; Electrodes; Expression Profiling; Fine needle aspiration biopsy; Fluorescence; Gene Expression; Gene Mutation; Genes; Genetic; Genomics; Goals; Gold; Healthcare; Kinetics; Label; Lasers; Measurement; Measures; Mediating; Medicine; Messenger RNA; Methods; Methylene blue; Microarray Analysis; Molecular; Natural regeneration; Nature; Noise; Nucleic Acids; Nucleotides; Outcome; Oxidation-Reduction; Pattern; Performance; Phase; Process; RNA; RNA-Directed DNA Polymerase; Reaction; Reading; Research; Scanning; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Speed; Spottings; Technology; Therapeutic; Time; Variant; base; gene discovery; interest; invention; logarithm; mRNA Expression; prognostic; statistics; stem; success; transcriptome

Methods for obtaining transcriptome data have revolutionized our understanding of biological processes and disease dynamics at the fundamental level, impacting health care on all three fronts: diagnostics, prognostics, and therapeutics. The compiling of transcriptome information, at one end of the spectrum leads to discovery of genes and mutations using, for example, the Sanger method and NetGen sequencing, and at the other end of the spectrum is the quantification of genetic expressions of known sequences using methods such as qPCR and microarrays. The proposed research pertains to the quantification of gene expression at few cell levels. Fundamental limitations of qPCR (the gold standard) and microarrays emerge from the inefficiencies and errors inherent to two necessary processes: cDNA synthesis by reverse transcriptase (RT) reaction, that requires more than 105 copies for reasonable efficiency, and subsequent PCR amplification, which may be prone to errors in exact replication. The goal of the proposed research is to develop a technology that eliminates these two processes and achieves at least an order of magnitude better sensitivity and better quality data in terms of self-consistency and normalization to accurately estimate relative expression levels of targeted mRNA. The expression level of 15 sequences from cell lines will be quantified simultaneously for this proof-of- principle study. The technology to be used is based on three principle steps: (i) targeting two unique sites of ~ 25 nucleotides on each mRNA of interest and exclusively separating the specifically bound target ssDNA sequences (TRID process); (ii) binding the ssDNA targets to a microarray mediated by electrochemical redox to obtain binding of ~ 1,800 molecules in 0.3 mL solution to microspots in less than 30 min at 100% specificity (EREB process); and (iii) reading the binding electrochemically at a responsivity of 0.4 zeptomole of probe- target binding to achieve a sensitivity of 10 attomolar and a dynamic range of five orders of magnitude (SEED process). It is expected that the time to result (TTR) after RNA extraction will be less than 3 hr. Specific Aim 1: Calibration of the SEED signal. The SEED signal for ssDNA targets for each mRNA from 10 aM to 10 pM in buffer will be measured to obtain calibration curves. The outcome will be the optimization of EREB to obtain copy numbers of all of the mRNA on a single chip. Specific Aim 2: qPCR study. Two cell lines of known dysregulation in genes will be cultured. The outcome will be mRNA expression of lysate of a known amount of cells by qPCR for all of the genes. Specific Aim 3: Technology verification study. TRID, EREB, and SEED will be performed at various dilutions of the same lysate solution used for qPCR. The outcome will be determination of the LOD and ENMC and the ability to measure at least 1.5-fold changes in copy number between the two cell lines and/or dilutions. Leveraging the high sensitivity and specificity, the long-term goal is to develop a quantitative microarray technology for gene expression of a few cells for applications such as single cell genomics, fine needle aspiration biopsy, and cell-free circulating nucleic acids.

获取转录组数据的方法已彻底改变了我们对生物过程的理解和 基本层面的疾病动态，影响所有三个方面的医疗保健：诊断，预后， 和治疗学。频谱的一端的转录组信息的编译导致发现 基因和突变使用，例如sanger方法和NetGen测序，在另一端 频谱是使用QPCR等方法对已知序列的遗传表达进行定量 和微阵列。拟议的研究与少数细胞水平上基因表达的定量有关。 QPCR（黄金标准）和微阵列的基本局限性来自效率低下和 两个必要过程固有的错误：通过逆转录酶（RT）反应cDNA合成， 需要超过105个副本才能合理效率和随后的PCR扩增，这可能是 容易出现精确复制的错误。拟议研究的目的是开发一种技术 消除这两个过程，并至少达到一个数量级的敏感性和质量更好 数据以自洽性和归一化来准确估计目标的相对表达水平 mRNA。从细胞系中的15个序列的表达水平将同时定量此证明 原则研究。要使用的技术基于三个主要步骤：（i）针对两个独特的站点〜 每个感兴趣的mRNA上的25个核苷酸，仅分离特定结合的靶标ssDNA 序列（TRID过程）; （ii）将ssDNA靶标与电化学氧化还原介导的微阵列结合 在不到30分钟的情况下，在100％特异性的情况下，在0.3 mL溶液中获得〜1,800分子的结合与微孔的结合 （EREB过程）； （iii）以0.4的探测的响应性来读取结合的电化学。 目标结合以达到10个attomolor的灵敏度和5个数量级的动态范围（种子 过程）。预计RNA提取后的结果（TTR）将小于3小时。具体目标 1：种子信号的校准。从上午10点到晚上10点，每个mRNA的ssDNA靶标的种子信号 在缓冲液中将测量以获得校准曲线。结果将是EREB的优化 复制单个芯片上所有mRNA的数字。特定目标2：QPCR研究。已知的两个细胞系 基因的失调将被培养。结果将是已知量的裂解物的mRNA表达 细胞通过qPCR用于所有基因。特定目标3：技术验证研究。 Trid，Ereb和种子将 以QPCR使用的相同裂解物溶液的各种稀释液进行。结果将是 LOD和ENMC的确定以及测量拷贝数至少1.5倍更改的能力 在两个细胞系和/或稀释液之间。利用高灵敏度和特异性，长期目标是 开发一种定量微阵列技术，用于一些用于应用的基因表达 单细胞基因组学，细针吸入活检和无细胞循环核酸。