Decoding pooled RNAi screens by means of barcode tiling arrays.
Decoding pooled RNAi screens by means of barcode tiling arrays.
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DOI：
10.1186/1471-2164-11-7
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发表时间：
2010-01-05
期刊：
影响因子：
4.4
通讯作者：
Hoheisel JD
中科院分区：
文献类型：
--
作者：
Boettcher M;Fredebohm J;Gholami AM;Hachmo Y;Dotan I;Canaani D;Hoheisel JD
最近，通过汇集的短发夹状RNA(ShRNAs)进行RNAi筛选已成为鉴定哺乳动物细胞中必需基因的有力工具。在过去的几年里，几个汇集在一起的大规模shRNA筛选已经确定了与癌细胞增殖有关的各种基因。所有这些研究都采用微阵列分析，利用shRNA的半发夹状序列或额外的shRNA相关的60nT条形码序列作为分子标签。在这里，我们描述了一种新的方法来解码汇集的RNAi屏幕，即条形码平铺阵列分析，并演示了如何使用该方法来精确量化池中单个shRNA的丰度。我们合成了具有6个重叠的25个核苷酸的长平铺探针的DNA微阵列，这些探针与每个shRNA表达构建体相关联的每个独特的60个核苷酸分子条形码序列互补。通过分析表达结构的稀释系列，我们展示了我们的方法如何允许量化池中的shRNA丰度，以及它如何明显优于通过shRNA的半发夹序列进行的常用分析。我们进一步展示了条形码平铺阵列如何用于从汇集的阴性选择筛选中预测单个shRNA的抗增殖效果。在305个shRNA池中，我们确定了28个候选shRNA，它们完全或部分地削弱了乳腺癌细胞系MDA-MB-231的生存能力。对11个shRNA表达构建体的子集的单独验证对细胞系的增殖具有潜在的抑制和非抑制作用，为条码拼接方法的准确性提供了进一步的证据。综上所述，我们提出了一种改进的方法，用于快速、定量和统计稳健地分析池RNAi筛选。我们的实验方法，加上商业上可用的慢病毒载体shRNA文库，有可能极大地促进发现癌症治疗的假定靶点以及药物毒性的增敏剂。
RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach. In summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.
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影响因子：
8.8
作者：
Canaani, D.
通讯作者：
Canaani, D.
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
影响因子：
48
作者：
Root, David E.;Hacohen, Nir;Sabatini, David M.
通讯作者：
Sabatini, David M.
影响因子：
14.9
作者：
Baum, M;Bielau, S;St채hler, PF
通讯作者：
St채hler, PF
影响因子：
30.8
作者：
THOMPSON, ME;JENSEN, RA;HOLT, JT
通讯作者：
HOLT, JT