Medical Biotechnology, VTT Technical Research Centre of Finland, 20521 Turku, Finland.
BMC Genomics. 2011 Mar 28;12:162. doi: 10.1186/1471-2164-12-162.
High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible.
Here, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells.
The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.
高通量 RNAi 筛选被广泛应用于生物学研究，但仍然昂贵，需要大量基础设施，并且许多测定法不适用于微孔板格式的高通量筛选。
在这里，我们描述了微型细胞点微阵列 (CSMA) 方法的优化，该方法促进了转染微阵列技术在不同 RNAi 分析中的应用。为了促进该方法的快速适应，我们使用一组 92 种贴壁细胞类型（包括原代人细胞）对该概念进行了测试。我们在对 492 个 GPCR 编码基因进行系统筛选，以研究其对培养的人类前列腺癌细胞生长和存活的影响中展示了该方法。
CSMA 方法有助于可重复地制备用于大规模基因敲低分析的高度平行细胞微阵列。这对于将基于细胞的功能遗传筛选扩展到包括更多 RNAi 构建体、允许组合 RNAi 分析、多参数表型读出或对许多不同细胞类型进行比较分析将是至关重要的。
