A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.
A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.
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DOI：
10.1089/adt.2005.3.401
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发表时间：
2005-09
影响因子：
1.8
通讯作者：
De-hua Yu;J. Chatterton;J. Bliesath;R. Sundaram;N. Ke;Vivian Nguy;B. Meyhack;F. Wong-Staal;
中科院分区：
文献类型：
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作者：
De-hua Yu;J. Chatterton;J. Bliesath;R. Sundaram;N. Ke;Vivian Nguy;B. Meyhack;F. Wong-Staal;
利用短干扰RNA瞬时转染肿瘤细胞，抑制肿瘤治疗基因的表达，是诱导肿瘤治疗表型（细胞凋亡、生长停滞等）的重要方法。用于癌症靶点验证。这些表型最初可以通过比色/荧光读数通过细胞存活来评估，例如，alamarBlue（Trek Diagnostic Systems，Cleveland，OH）和WST-1。然而，瞬时转染存在固有的问题-变化的毒性，不一致的转染效率，以及其他细胞特异性的决定因素-这有助于低信噪比的测定，使测定无效，特别是当应用于高通量筛选（HTS）多路复用不同的细胞。本报告描述了一种使用报告基因作为96孔格式中常规存活读数的“归一化替代物”的方法。在这种方法中，只有转染的存活细胞产生报告活性，并且排除了与瞬时转染相关的许多变量。将组成型表达的报告基因（荧光素酶或LacZ）表达盒与专门设计的RNA干扰（RNAi）载体（或为此的转基因）一起共转染到细胞中沿着。然后原位定量液体培养物或96孔形式的软琼脂培养物中的报告物活性。简化了RNAi载体的构建，使其能够适应96孔格式。我们的数据表明，存活率的相对报告读数与转染效率和细胞毒性无关。信噪比显著增加，特别是对于低转染效率的细胞。该测定是通用的和稳健的，并且可以应用于用于癌症靶点鉴定和验证的多重HTS。
Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.