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

获得转录组数据的方法彻底改变了我们对生物过程和 基础层面的疾病动态，在所有三条战线上影响卫生保健：诊断、预后、 和治疗学。转录组信息的编辑，在光谱的一端导致发现 例如，使用桑格方法和NetGen测序的基因和突变，以及在 谱是使用诸如qPCR等方法对已知序列的遗传表达进行量化 和微阵列。这项拟议的研究涉及在少数细胞水平上对基因表达进行量化。 定量聚合酶链式反应(金标准)和微阵列的根本局限性来自于低效率和 两个必要的过程所固有的错误：通过逆转录酶(RT)反应合成cDNA，即 需要超过105个拷贝才能获得合理的效率，并随后进行PCR扩增，这可能是 在精确复制中容易出错。这项拟议的研究的目标是开发一种 消除了这两个过程，并实现了至少一个数量级的更高的灵敏度和更好的质量 在自我一致性和归一化方面的数据，以准确估计目标的相对表达水平 MRNA.来自细胞系的15个序列的表达水平将同时被量化，以证明- 原则性研究。将使用的技术基于三个主要步骤：(I)瞄准两个独特的地点~ 在每个感兴趣的mRNA上有25个核苷酸，并唯一地分离特异结合的目标单链DNA 序列(TRID过程)；(Ii)通过电化学氧化还原将单链DNA靶标与微阵列结合 在不到30分钟的时间内，以100%的特异度获得0.3mL溶液中的~1800个分子与微斑点的结合 (ereb过程)；以及(Iii)以0.4zeptomole的探针响应率电化学读取结合- 目标结合以实现10个大气压的灵敏度和五个数量级(种子)的动态范围 过程)。预计RNA提取后的结果时间(Ttr)将不到3小时。特定目标 1：种子信号的校准。从上午10点到晚上10点，每个mRNA的单链DNA靶标的种子信号 在缓冲液中进行测量，得到校准曲线。最终的结果将是eREB的优化，以获得 在单个芯片上复制所有信使核糖核酸的编号。具体目标2：定量聚合酶链式反应研究。已知的两个细胞系 基因调节失调将会被培养出来。结果将是已知量的裂解物的mRNA表达 对所有基因进行定量聚合酶链式反应。具体目标3：技术验证研究。Trid、Ereb和Seed Will 在不同稀释度下进行定量聚合酶链式反应所用的同一裂解液。结果将是 LOD和ENMC的确定以及测量拷贝数至少1.5倍变化的能力 在两个细胞系和/或稀释液之间。利用高度的敏感性和特异性，长期目标是 为了开发用于少数细胞基因表达的定量微阵列技术，例如 单细胞基因组学、细针活检和无细胞循环核酸。

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.