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

基本信息

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
项目类别：
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

项目摘要

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彻底改变了我们对生物过程和基本层面的疾病动态，从三个方面影响医疗保健：诊断、预后、和治疗方法。转录组信息的编译，一方面导致发现使用例如 Sanger 方法和 NetGen 测序的基因和突变，以及在另一端谱图是使用 qPCR 等方法对已知序列的基因表达进行量化和微阵列。拟议的研究涉及少数细胞水平上基因表达的量化。 qPCR（黄金标准）和微阵列的基本局限性源于效率低下和两个必要过程固有的错误：通过逆转录酶 (RT) 反应合成 cDNA，即需要超过 105 个拷贝才能达到合理的效率，以及随后的 PCR 扩增，这可能是精确复制时容易出现错误。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高灵敏度和更好质量数据的自我一致性和标准化，以准确估计目标的相对表达水平 mRNA。为了证明这一点，将同时对来自细胞系的 15 个序列的表达水平进行定量原理研究。所使用的技术基于三个原则步骤：（i）针对〜的两个独特位点每个感兴趣的 mRNA 上有 25 个核苷酸，并专门分离特异性结合的目标 ssDNA 序列（TRID 过程）； (ii) 通过电化学氧化还原将 ssDNA 靶标结合到微阵列上在 30 分钟内以 100% 特异性将 0.3 mL 溶液中的约 1,800 个分子与微点结合（EREB 过程）； (iii) 以 0.4 Zeptomole 探针的响应度以电化学方式读取结合目标结合以实现 10 阿托摩尔的灵敏度和 5 个数量级的动态范围 (SEED 过程）。预计 RNA 提取后获得结果的时间 (TTR) 将少于 3 小时。具体目标 1：SEED 信号的校准。上午 10 点至下午 10 点期间每个 mRNA 的 ssDNA 目标的 SEED 信号将测量缓冲液中的溶液以获得校准曲线。结果将是 EREB 的优化以获得单个芯片上所有 mRNA 的拷贝数。具体目标 2：qPCR 研究。已知的两种细胞系基因失调将被培养。结果将是已知量的裂解物的 mRNA 表达通过 qPCR 对细胞中的所有基因进行检测。具体目标3：技术验证研究。 TRID、EREB 和 SEED 将可以在用于 qPCR 的相同裂解物溶液的不同稀释度下进行。结果将是确定 LOD 和 ENMC，并能够测量至少 1.5 倍的拷贝数变化两种细胞系和/或稀释度之间的差异。利用高灵敏度和特异性，长期目标是开发一种用于一些细胞基因表达的定量微阵列技术，用于诸如单细胞基因组学、细针抽吸活检和无细胞循环核酸。