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siRNA/ miRNA高通量筛选
日 12:05&&&&&&字体：【&&】
1.背景介绍
RNAi技术是一种新型的基因沉默工具，可简单、快速、特异地分析特定基因的细胞功能。其工作原理如下：在内源蛋白复合物（RISC）的作用下，双链RNA分子可以通过碱基互补配对原则而降解目标靶基因的信使RNA（mRNA），从而达到沉默靶基因表达水平的目的。在全基因范围，筛选与确认特定细胞行为的功能基因已成为药物化学、生命科学研究已经应用的常规手段。基于SiRNA全基因组文库的高通量筛选技术，可平行分析成千上万条基因的功能，大大加快研究进程，从而能迅速、全面地鉴定出细胞过程或信号传导途径的相关候选基因，以及疾病相关的药物靶基因，保证研究成果更为系统、新颖。 2.常规孔板筛选办法
常规的siRNA筛选是基于384或96微孔板，采用一般转染siRNA的方法或者反式转染方法将siRNA导入细胞进行高通量的细胞功能筛选。其中反式转染法由于操作方便，时间较短，常被高通量筛选采用。简单讲，预先将siRNA文库里各条siRNA分别与转染试剂混合，转移到384微孔板中，然后将培养好的细胞接种到微孔板里，细胞被转染siRNA。经过48-72小时的培养，蛋白表达水平沉默后，通过高通量的微孔板读数仪或高通量的扫描显微镜读出孔板里的细胞信号。若这些细胞信号与参照细胞相比发生了变化，则认为相应的siRNA与研究的细胞过程相关，从而鉴定出相关的候选基因。
传统siRNA筛选示意图
微孔板筛选虽然简单明了，使用方便，适合于100-500个基因范围的筛选。但是当筛选库容量增大时，孔板筛选的成本将大大提高。比如，全基因组2万个基因，若重复三次筛选，需做6万次转染、分析，因此使用的siRNA、转染试剂、以及后续检测用的抗体和底物需要量特别大。 3.芯片筛选技术
除了传统的孔板筛选服务外，我们针对大规模筛选需求，成功开发出先进的SAMcell（Self-Assemble Microcell）细胞芯片技术，在玻璃盖玻片上，细胞自组装形成细胞岛点阵，极大地提高了筛选通量。同时采用反向转染技术，简化了筛选过程，同时大大减少筛选中使用的SiRNA和相应试剂的用量。
SAMcell技术示意图:在芯片上点印siRNA或miRNA，接种细胞后，细胞自组装形成细胞岛点阵，同时siRNA或miRNA自动转染到对应的细胞小岛，最后通过高内涵图像扫描系统成像，分析实验结果。
细胞岛间无交叉污染：在20x20mm的盖玻片上，细胞在自组装形成点阵的同时转染表达GFP的质粒，细胞小岛间隔形成荧光点阵，而相邻的细胞小岛（没有转染GFP质粒）则没有荧光信号。 技术特点：
1. 筛选通量极大提高。
2.操作简单，只需要像传统的细胞培养操作一样加入细胞，细胞自组装自转染，使得大规模筛选十分方便。
3.验平行性好,稳定性提高100倍，所有细胞小岛在同样的培养环境，避免384孔板间实验条件差别。
4.成本是现有筛选技术的十分之一,&&SiRNA、转染试剂及检测抗体用量都大大降低。
SAM cell技术已经大量应用于SiRNA基因文库及化合物库高通量筛选。 4.筛选设备
拥有高通量液体工作站、高内涵成像仪、微孔板读数仪、酶标仪、高通量细胞分装以及清洗仪先进的全套高通量筛选设备
高通量液体工作站高通量图像扫描显微镜
微孔板读数仪dispenser 5.目前提供如下规模文库筛选 1) 全基因组siRNA文库（约21585genes）
2) 人类全miRNA文库及inhibitor（904条） 3) 细胞周期调节siRNA&&文库（131genes） 4) 去泛素化酶siRNA文库（127genes） 5) 磷酸化酶文库（257genes） 6) 细胞因子受体siRNA文库（319genes） 7) G蛋白偶联受体siRNA文库(516genes)
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【求助】shRNA文库结合下一代测序技术筛选合成致死因子
【求助】shRNA文库结合下一代测序技术筛选合成致死因子
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问题已解决悬赏丁当:2
各位战友，有谁了解shRNA文库结合下一代测序技术筛选合成致死因子的原理的，英文文献看不懂，求中文解释。
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
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Can you uploa
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
分享到哪里？
Can you upload the original research paper or show the link? Thanks!
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
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mpark Can you upload the original research paper or show the link? Thanks!1.Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNAscreens.2.A comprehensive platform for highly multiplexed mammalian functional geneticscreens3.Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen4.Identification of unique sensitizing targets for anti-inflammatory CDDO-Me in metastatic melanoma by a large-scale synthetic lethal RNAi screening5.Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer主要是涉及到这几篇文献，shRNA文库结合下一代测序技术筛选仅在第一篇有详细介绍，但是没有用于筛选合成致死因子。后面几篇是用siRNA或者shRNA文库结合基因微阵列筛选的。我主要是不理解shRNA文库转染细胞株后，是使用什么条件筛选出，然后怎么测序分析出来合成致死因子的基因的。
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
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Figure 1The picture is from Hendrik J Kuiken, Roderick L Beijersbergen paper published on Future Oncol. ):. In the paper, it is said that &T he development of vector systems expressing shRNAs, which are processed in the cell to siRNAs, have provided a tool for long-term experiments.& In screening, &in contrast to siRNA libraries, shRNA libraries can be used in a pooled format (Figure 1). In this approach large numbers of shRNA vectors are combined and used to infect a single, large population of cells. After selection, the integrated shRNA cassettes can be recovered by PCR on genomic DNA and the relative abundance of each individual shRNA can be determined by hybridization to DNA microarrays or .
The barcode technology has been applied successfully for positive selection screens and, to a lesser extent, in negative selection screens.&Figure 2From:,
doi: 10.1016/j.febslet.. It is said that &Overview of a pooled shRNA screen set-up. Cell lines (A and B) are infected with short hairpin RNA libraries targeting thousands of gene products by RNAi (Figure 2). Cells are cultured to allow the depletion of those containing shRNAs that target essential genes. Genomic DNA is isolated and the vectors are quantified using so-called barcode sequences (short stretches of DNA) that are unique for each shRNA vector. By comparing the genes that are required in one cell line but not the other by custom micro-array or deep sequencing, potential synthetic interactions can be identified.&In the attachment is a published paper regarding approach of synthetic lethality shRNA library screen plus deep sequencing.
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
分享到哪里？
mpark Figure 1The picture is from Hendrik J Kuiken, Roderick L Beijersbergen paper published on Future Oncol. ):. In the paper, it is said that &T he development of vector systems expressing shRNAs, which are processed in the cell to siRNAs, have provided a tool for long-term experiments.& In screening, &in contrast to siRNA libraries, shRNA libraries can be used in a pooled format (Figure 1). In this approach large numbers of shRNA vectors are combined and used to infect a single, large population of cells. After selection, the integrated shRNA cassettes can be recovered by PCR on genomic DNA and the relative abundance of each individual shRNA can be determined by hybridization to DNA microarrays or .
The barcode technology has been applied successfully for positive selection screens and, to a lesser extent, in negative selection screens.&Figure 2From:,
doi: 10.1016/j.febslet.. It is said that &Overview of a pooled shRNA screen set-up. Cell lines (A and B) are infected with short hairpin RNA libraries targeting thousands of gene products by RNAi (Figure 2). Cells are cultured to allow the depletion of those containing shRNAs that target essential genes. Genomic DNA is isolated and the vectors are quantified using so-called barcode sequences (short stretches of DNA) that are unique for each shRNA vector. By comparing the genes that are required in one cell line but not the other by custom micro-array or deep sequencing, potential synthetic interactions can be identified.&In the attachment is a published paper regarding approach of synthetic lethality shRNA library screen plus deep sequencing. Thanks so much！！
回复：【求助】shRNA文库结合下一代测序技术筛选合成致死因子
分享到哪里？
mpark Figure 1The picture is from Hendrik J Kuiken, Roderick L Beijersbergen paper published on Future Oncol. ):. In the paper, it is said that &T he development of vector systems expressing shRNAs, which are processed in the cell to siRNAs, have provided a tool for long-term experiments.& In screening, &in contrast to siRNA libraries, shRNA libraries can be used in a pooled format (Figure 1). In this approach large numbers of shRNA vectors are combined and used to infect a single, large population of cells. After selection, the integrated shRNA cassettes can be recovered by PCR on genomic DNA and the relative abundance of each individual shRNA can be determined by hybridization to DNA microarrays or .
The barcode technology has been applied successfully for positive selection screens and, to a lesser extent, in negative selection screens.&Figure 2From:,
doi: 10.1016/j.febslet.. It is said that &Overview of a pooled shRNA screen set-up. Cell lines (A and B) are infected with short hairpin RNA libraries targeting thousands of gene products by RNAi (Figure 2). Cells are cultured to allow the depletion of those containing shRNAs that target essential genes. Genomic DNA is isolated and the vectors are quantified using so-called barcode sequences (short stretches of DNA) that are unique for each shRNA vector. By comparing the genes that are required in one cell line but not the other by custom micro-array or deep sequencing, potential synthetic interactions can be identified.&In the attachment is a published paper regarding approach of synthetic lethality shRNA library screen plus deep sequencing.您好！您回复的帖子里面第二幅图上有句话“Grow cells to allow depletion of required shRNAs”我有点不明白，您能给我解释一下吗？非常感谢！
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