High Specificity MicroRNA Microarray Analysis without PCR for Cancer Screening and Research

 描述（由申请人提供）：最近的研究，例如关于胰腺癌的研究，表明microRNA（miRNA）是几种类型癌症的有效生物标志物，其中超过100种被鉴定为癌基因、肿瘤抑制因子以及癌症干细胞和转移的调节剂。miRNA谱具有在临床体征出现之前以高特异性在癌症的早期诊断中高度有效的潜力。目前用于miRNA测序的技术，定量逆转录-聚合酶链反应（qRT-PCR），难以多重化并且太昂贵而不能用于筛选。拟议的研究将开发一种使用微阵列进行定量miRNA分析的技术，该技术成本低得多，易于多重化。然而，微阵列具有挑战：（a）由于非特异性结合导致的统计学差，miRNA的小尺寸使得推断不可靠;（B）来自相同pre-miRNA家族的序列中的一到几个核苷酸（nt）变异难以辨别，（c）用于制备cDNA的小miRNA的RT-PCR不是直接的，和（d）PCR通过夸大相对浓度较大的miRNA序列的扩增而使分布偏斜。R21研究的目标是开发一种微阵列方法，通过测量探针-靶标结合来分析miRNA序列，而无需PCR，这种结合对非特异性结合是“盲”的。该信号将定量区分完美结合（PM）、单核苷酸错配（1 MM）和大小异质性。这项概念验证研究将重点关注相同序列的合成和血液来源的miRNA。Saraf实验室开发的一种方法，可以通过简单的激光扫描电化学“读取”单片电极上的微阵列点。双电层扫描静电计（SEED）可以检测到小于1阿摩尔（amoles）的固定化探针-靶结合，并区分PM和1 MM;非特异性结合产生最小信号。SEED的0.1aMole响应度将利用额外的创新来提高结合效率，以实现至少0.05 nM的检测限（LOD），需要0.2 ng miRNA。该研究将被组织成三个具体目标：1）使用合成的miRNA量化SEED性能，2）定量分析合成的miRNA的混合物，以及3）使用SEED分析来自胰腺癌患者和健康对照的血清样品。SEED是一种潜在的变革性技术，它将利用快速增长的miRNA生物标志物知识库和经过验证的电化学转导方法，在最小背景下检测特异性结合，而无需使用标记。该技术的范例是，它在硬件检测水平上而不是在实验设计水平上解决了微阵列中的背景问题。如果成功，该团队将通过R33使用特定癌症的生物标本来验证该方法，以启动筛选技术和假设驱动的R 01研究。

 DESCRIPTION (provided by applicant): Recent studies, such as those on pancreatic cancer, indicate that microRNA (miRNA) are effective biomarkers for several types of cancer, with over 100 of them identified that act as oncogenes, tumor suppressors, and modulators of cancer stem cells and metastasis. MiRNA profiles have the potential to be highly effective in the early diagnosis of cancer at high specificity before clinical signs emerge. Current technology for miRNA sequencing, quantitative reverse transcribe-polymerase chain reaction (qRT-PCR), is difficult to multiplex and too expensive to be used for screening. The proposed research will develop a technology for quantitative miRNA profiling using microarrays that are considerably less expensive and easy to multiplex. Microarrays, however, have challenges: (a) the small size of miRNA makes the inference unreliable due to poor statistics caused by nonspecific binding; (b) the one to few nucleotide (nt) variation in the sequence from the same family of pre-miRNA is difficult to discern, (c) RT-PCR of small miRNAs used to make cDNA is not straightforward, and (d) PCR skews the distribution by exaggerating the amplification of miRNA sequences that are larger in relative concentration. The goal of the proposed R21 research is to develop a microarray method for profiling a miRNA sequence without PCR by measuring probe-target binding that is "blind" to nonspecific binding. The signal will quantitatively distinguish between perfect binding (PM), single nucleotide mismatch (1MM), and size heterogeneity. This proof-of-concept study will focus on synthetic and blood derived miRNA of the same sequence. A method, developed in Saraf's lab, can electrochemically "read" microarray spots on a monolith electrode by simply scanning a laser. Scanning Electrometer for Electrical Double-layer (SEED) can detect less than 1 atto-moles (amoles) of immobilized probe-target binding and differentiate between PM and 1MM; and nonspecific binding produces minimal signal. The 0.1aMole responsivity of SEED will be leveraged using an additional innovation to enhance binding efficiency to achieve a limit of detection (LOD) of at least 0.05 nM, requiring 0.2 ng miRNA. The study will be organized into three specific aims: 1) SEED performance will be quantified using synthetic miRNA, 2) mixtures of synthetic miRNA will be quantitatively analyzed, and 3) serum samples from pancreatic cancer patients and healthy controls will be analyzed using SEED. SEED is a potentially transformative technology that will leverage the rapidly growing knowledge base of miRNA biomarkers and a proven electrochemical transduction method for detecting specific binding without using labels at minimal background. The paradigm of the technology is that it addresses the background issue in microarrays at the hardware detection level rather than at the design-of-experiment level. If successful, the team will validate the method via an R33 using biospecimens for specific cancers to initiate translation to a screening technology and hypothesis-driven R01 research.