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

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.

获得转录组数据的方法已经彻底改变了我们对生物过程的理解， 在基础层面的疾病动态，影响所有三个方面的卫生保健：诊断，免疫学， 和治疗学。转录组信息的编译，在光谱的一端导致发现 使用例如桑格方法和NetGen测序，在基因和突变的另一端， 光谱是使用诸如qPCR的方法对已知序列的遗传表达进行定量 和微阵列。拟议的研究涉及在少数细胞水平上定量基因表达。 qPCR（金标准）和微阵列的基本局限性来自于效率低下， 两个必要过程固有的错误：通过逆转录酶（RT）反应的cDNA合成， 需要超过105个拷贝以获得合理的效率，以及随后的PCR扩增， 在精确复制时容易出错。拟议研究的目标是开发一种技术， 消除了这两个过程，并实现了至少一个数量级的更好的灵敏度和更好的质量 数据的自洽性和标准化，以准确估计靶向的相对表达水平。 mRNA。将同时定量来自细胞系的15个序列的表达水平，以用于该验证。 原则研究。所使用的技术基于三个原则步骤：（i）靶向~的两个独特位点， 每个感兴趣的mRNA上的25个核苷酸，并专门分离特异性结合的靶ssDNA 序列（TRID过程）;（ii）通过电化学氧化还原介导将ssDNA靶结合到微阵列 在100%特异性下，在不到30分钟的时间内，获得0.3 mL溶液中约1，800个分子与微点的结合 (EREB方法）;和（iii）在0.4泽托摩尔探针的响应度下，以电化学方式阅读结合。 靶结合以实现10阿摩尔的灵敏度和5个数量级的动态范围（SEED 过程）。预计RNA提取后的结果时间（TTR）将小于3小时。 1：校准种子信号。ssDNA的SEED信号靶向10 aM至10 pM的每种mRNA 将测量缓冲液中的浓度以获得校准曲线。结果将是EREB的优化，以获得 所有mRNA的拷贝数。具体目标2：qPCR研究。两种已知的 基因的失调将被培养。结果将是已知量的细胞裂解物的mRNA表达。 通过qPCR检测所有基因的细胞。具体目标3：技术验证研究。TRID、EREB和SEED将 在用于qPCR的相同裂解物溶液的各种稀释度下进行。结果会 LOD和ENMC的测定以及测量拷贝数至少1.5倍变化的能力 两种细胞系和/或稀释液之间的差异。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，用于一些细胞的基因表达， 单细胞基因组学、细针穿刺活检和无细胞循环核酸。