Multiplexed Gene Assays by Microtiter Plate Microarrays

描述（由申请人提供）：药物发现行业正面临越来越大的压力，需要根据其疗效和特异性快速优先考虑候选化合物。虽然传统的基于芯片的基因表达微阵列可以提供重要的见解，样品格式和吞吐量的考虑排除了更广泛的使用。基于微量滴定板的微阵列（MPMA）系统可以直接集成到现有的药物发现过程中，并将解决高样品通量多重基因表达测定的需求。抗病毒药物发现公司Apath LLC和基于微阵列的微量滴定板公司Proteoprotein提出合作开发适合药物发现应用（如毒物鉴定）的多重基因表达测定。Proteoprotein已经开发出了每个微量滴定孔可点样多达144个寡核苷酸探针的系统，Apath已经充分表征了病毒复制子系统和细胞毒性数据，这些数据有助于检测验证。我们最初计划通过使用纯化的体外转录本和修改探针沉积、杂交和cDNA靶标记条件来优化检测灵敏度和重现性。接下来，我们将证明MPMA可以忠实地再现剂量反应效应，通过集中在充分表征的干扰素介导的病毒复制子RNA的抑制。最后，我们将通过对表征良好的化合物组进行盲筛选并将结果与标准细胞毒性试验中获得的结果进行比较，证明MPMA在识别活性和毒性化合物方面的实用性。

DESCRIPTION (provided by applicant): The drug discovery industry is under increasing pressure to rapidly prioritize compound candidates based on their efficacy and specificity. Although traditional chip based gene expression microarrays can provide significant insights, sample format and throughput considerations preclude more widespread use. Microtiter plate based microarray (MPMA) systems can be directly integrated into the existing drug discovery process and would address the need for high sample throughput multiplexed gene expression assays. Apath LLC, an antiviral drug discovery company, and ProteoPlex, a microtiter plate based microarray company propose a collaboration to develop a multiplexed gene expression assay suitable for drug discovery applications such as toxicant identification. ProteoPlex has developed systems for spotting up to 144 oligonucleotide probes per microtiter well and Apath has well characterized viral replicon systems and cytotoxicity data that facilitate assay validation. We initially plan to optimize assay sensitivity and reproducibility through use of purified in vitro transcripts and modification of the probe deposition, hybridization, and cDNA target labeling conditions. We will next demonstrate that the MPMA can faithfully reproduce dose response effects by focusing on the well characterized interferon mediated inhibition of viral replicon RNAs. Finally, we will demonstrate the utility of the MPMA in identification of active and toxic compounds by performing blinded screens on panels of well-characterized compounds and comparing the results to those obtained in standard cytotoxicity assays.