Name: high-throughput dna plasmid multiplexing and transfection using acoustic nanodispensing technology
Uploaded: 2019-08-08T13:00:00
Description: Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

作者披露了对本文的研究、作者和/或出版物的下列财政支持:因塞姆、里尔大学、里尔巴斯德研究所、北方理事会以及PRIM-HCV1和2(Recherche)(联邦国际医药协会、国家重建局(ANR-10-EQPX-04-01)、Feder(12001407(D-AL)和欧洲共同体(ERC-STG INTRACELLTB n=260901)。作者要感谢S.Mouru博士、B.Villemagne博士、R.Ferru-Clément博士和H.Groult博士对手稿的批判性回顾和更正。

1. 提前准备
<ol><li>准备蠕动液体处理程序 注:对于协议的稀释剂和单元分配步骤,必须准备专用程序,同时考虑到使用板的点胶头高度和步骤意图。<ol>
<li>对于 1 μL 稀释剂点胶步骤,安装 1 μL 盒式磁带,并准备步骤 1.1.1.1 和 1.1.1.2 中所述的设置程序。<ol>
<li>将流速参数调整为"高",以获得最佳吞吐量,因为此步骤中预计不会造成生物材料损坏。将分配高度调整到 9.6 mm(根据所?...

<ol>
	<li>Colin, B., Deprez, B., Couturier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+DNA+Plasmid+Transfection+Using+Acoustic+Droplet+Ejection+Technology.">High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.</a> SLAS Discovery: Advancing Life Sciences R &#38; D. , 2472555218803064 (2018).</li><li>Mirus Bio. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimising Transfection Performance. , (2019).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors Influencing Transfection Efficiency | Thermo Fisher Scientific - FR. , (2019).</li><li>Boussif, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (16), 7297-7301 (1995).</li><li>Figueroa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanistic+investigation+exploring+the+differential+transfection+efficiencies+between+the+easy-to-transfect+SK-BR3+and+difficult-to-transfect+CT26+cell+lines.">A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.</a> Journal of Nanobiotechnology. 15 (1), 36 (2017).</li><li>Kirchenbuechler, I., Kirchenbuechler, D., Elbaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+cationic+lipid-based+transfection+and+cell+division.">Correlation between cationic lipid-based transfection and cell division.</a> Experimental Cell Research. 345 (1), 1-5 (2016).</li><li>Zhang, Z., Qiu, S., Zhang, X., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+DNA+electroporation+for+primary+human+T+cell+engineering.">Optimized DNA electroporation for primary human T cell engineering.</a> BMC Biotechnology. 18 (1), 4 (2018).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+activity+and+the+mechanism+of+pDNA-complexes+based+on+the+hybrid+of+low-generation+PAMAM+and+branched+PEI-1.8k.">Transfection activity and the mechanism of pDNA-complexes based on the hybrid of low-generation PAMAM and branched PEI-1.8k.</a> Molecular bioSystems. 9 (12), 3175-3186 (2013).</li><li>Bos, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+semi-automated+high+throughput+transient+transfection+system.">Development of a semi-automated high throughput transient transfection system.</a> Journal of Biotechnology. 180, 10-16 (2014).</li><li>Colosimo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+and+expression+of+foreign+genes+in+mammalian+cells.">Transfer and expression of foreign genes in mammalian cells.</a> BioTechniques. 29 (2), 314-318 (2000).</li><li>Villa-Diaz, L. G., Garcia-Perez, J. L., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+transfection+efficiency+of+human+embryonic+stem+cells+by+the+incorporation+of+DNA+liposomes+in+extracellular+matrix.">Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.</a> Stem Cells and Development. 19 (12), 1949-1957 (2010).</li><li>Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reverse transfection method. , WO2001020015A1 (2001).</li><li>Raymond, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+polyethylenimine-mediated+transfection+process+for+large-scale+and+high-throughput+applications.">A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications.</a> Methods (San Diego, CA). 55 (1), 44-51 (2011).</li><li>Junquera, E., Aicart, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+gene+therapy+to+deliver+nucleic+acids+with+multivalent+cationic+vectors.">Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors.</a> Advances in Colloid and Interface Science. 233, 161-175 (2016).</li><li>Woodruff, K., Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High-Throughput+Microfluidic+Platform+for+Mammalian+Cell+Transfection+and+Culturing.">A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing.</a> Scientific Reports. 6, 23937 (2016).</li><li>Vasileiou, T., Foresti, D., Bayram, A., Poulikakos, D., Ferrari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Contactless+Biology:+Acoustophoretic+DNA+Transfection.">Toward Contactless Biology: Acoustophoretic DNA Transfection.</a> Scientific Reports. 6, 20023 (2016).</li><li>Hadimioglu, B., Stearns, R., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+Liquids+with+Sound:+The+Physics+of+Acoustic+Droplet+Ejection+for+Robust+Laboratory+Automation+in+Life+Sciences.">Moving Liquids with Sound: The Physics of Acoustic Droplet Ejection for Robust Laboratory Automation in Life Sciences.</a> Journal of Laboratory Automation. 21 (1), 4-18 (2016).</li><li>Grant, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> Journal of Biomolecular Screening. 14 (5), 452-459 (2009).</li><li>Sackmann, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+That+Enable+Accurate+and+Precise+Nano-+to+Milliliter-Scale+Liquid+Dispensing+of+Aqueous+Reagents+Using+Acoustic+Droplet+Ejection.">Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.</a> Journal of Laboratory Automation. 21 (1), 166-177 (2016).</li><li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chemical Society Reviews. 38 (10), 2887-2921 (2009).</li><li>Zielinski, D., Gordon, A., Zaks, B. L., Erlich, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPipet:+sample+handling+using+a+tablet.">iPipet: sample handling using a tablet.</a> Nature Methods. 11 (8), 784-785 (2014).</li><li>Brunner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+dependence+of+gene+transfer+by+lipoplex,+polyplex+and+recombinant+adenovirus.">Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.</a> Gene Therapy. 7 (5), 401-407 (2000).</li><li>Nii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell-State+Culture+of+Human+Pluripotent+Stem+Cells+Increases+Transfection+Efficiency.">Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.</a> BioResearch Open Access. 5 (1), 127-136 (2016).</li><li>Noonan, D. J., Henry, K., Twaroski, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High-Throughput+Mammalian+Cell-Based+Transient+Transfection+Assay.">A High-Throughput Mammalian Cell-Based Transient Transfection Assay.</a> Signal Transduction Protocols. 284, 051-066 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transfection | TransIT Transfection Reagents | Mirus Bio. , (2015).</li><li>American Type Culture Collection. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> General protocol for transfection of stem cells, primary cells, and continuous cell lines with ATCC TransfeX Transfection Reagent. , (2017).</li><li>American Type Culture Collection. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transfection Reagents for Nucleic Acid Transfer into ATCC Cells. , (2017).</li><li>de Los Milagros Bassani Molinas, M., Beer, C., Hesse, F., Wirth, M., Wagner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+transient+transfection+process+of+HEK-293+suspension+cells+for+protein+production+by+nucleotide+ratio+monitoring.">Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.</a> Cytotechnology. 66 (3), 493-514 (2014).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FuGENE&#174; 6 Transfection Reagent. , (2019).</li><li>Olden, B. R., Cheng, Y., Yu, J. L., Pun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+polymers+for+non-viral+gene+delivery+to+human+T+cells.">Cationic polymers for non-viral gene delivery to human T cells.</a> Journal of Controlled Release: Official Journal of the Controlled Release Society. 282, 140-147 (2018).</li><li>Park, E., Cho, H. B., Takimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+gene+delivery+into+adipose-derived+stem+cells:+transfection+of+cells+in+suspension+with+the+use+of+a+nuclear+localization+signal+peptide-conjugated+polyethylenimine.">Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.</a> Cytotherapy. 17 (5), 536-542 (2015).</li><li>Wood, R. W., Loomis, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physical+and+biological+effects+of+high-frequency+sound-waves+of+great+intensity.">The physical and biological effects of high-frequency sound-waves of great intensity.</a> The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science. 4 (22), 417-436 (1927).</li><li>Mamat, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Endotoxin+at+the+Source+-+A+Novel+Competent+Cell+Line+with+Modified+Lipopolysaccharide+for+Low-Endotoxin+Plasmid+Production.">Eliminating Endotoxin at the Source - A Novel Competent Cell Line with Modified Lipopolysaccharide for Low-Endotoxin Plasmid Production.</a> , (2014).</li><li>Ivanova, N. V., Kuzmina, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+dry+DNA+storage+and+shipment+at+room+temperature.">Protocols for dry DNA storage and shipment at room temperature.</a> Molecular Ecology Resources. 13 (5), 890-898 (2013).</li><li>Lesnick, J., Lejeune-Dodge, A., Ruppert, N., Jarman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Precision Cell Dispensing with the Labcyte Echo&#174; Liquid Handler. , (2017).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+public+genome-scale+lentiviral+expression+library+of+human+ORFs.">A public genome-scale lentiviral expression library of human ORFs.</a> Nature Methods. 8 (8), 659-661 (2011).</li><li>Peng, J., Zhou, Y., Zhu, S., Wei, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+in+mammalian+cells+using+the+CRISPR-Cas9+system.">High-throughput screens in mammalian cells using the CRISPR-Cas9 system.</a> The FEBS journal. 282 (11), 2089-2096 (2015).</li></ol>

为给定细胞系建立和优化精确的高通量转染方法需要科学家遵循本节中描述的一些关键参数。我们强烈鼓励从整个协议中的建议值开始,因为这些针对 HeLa 细胞优化的设置也证明对 HEK 细胞有效。然而,由于最佳参数可能取决于细胞系和转染试剂,因此可以通过改变细胞数量、稀释体积、总DNA量和转染试剂的性质、浓度,甚至体积来定义最佳条件。在HeLa细胞1优化该协议期间。
<p cla...

这里介绍的方法详细介绍了如何在384孔板中使用声基液体纳米分配器在高通量的哺乳动物细胞中执行DNA质粒多路复用和转染,即使是对于该领域的非专家也是如此。最近公布的方法1允许在一个实验中执行多达384个独立的质粒DNA复用和转染条件,在不到1小时。转染细胞群中的共转染。此协议使转染更容易,因为大多数繁琐、耗时且容易出错的步骤现在都是软件驱动的(有关概述,请参阅图1)。已作出进一步努力,开发专用工具,以提高易用性,同时避免整个过程中的人为错误,并促进成功的转染,即使是外地的非专家。所述协议包括一个"用户友好"的宏电子表格,我们开发了该电子表格,以便管理 384 个独立的转染条件,每个孔中最多具有四个质粒的多路复用可能性。宏自动生成源板的模板,以从起始库存解决方案加载预期的 DNA 质粒体积,并在已输入的实验设计后启动纳米分配器软件所需的文件。由于在384孔源盘中手动分配DNA是繁琐且容易出错的,我们还开发了一个基于平板电脑的专用应用程序,在根据模板分配DNA溶液的同时指导用户。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59570/59570fig1v3.jpg"> 图1:实验工作流。最佳自动化高通量反向转染协议(从实验设计到定制生物测定)的原理表示。手动步骤由手符号指示,每个步骤的大致时间写在红色框中。<a href="https://www.jove.com/files/ftp_upload/59570/59570fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
许多基于细胞的实验从质粒DNA转染开始,即使许多专用试剂已经和仍在开发中,以提高转染效率和/或简化程序,仍有许多工作要做2,3,4.DNA质粒细胞转染涉及几个步骤,以达到高效率,如初始复杂接受,内皮逃逸,和细胞质传输到细胞核5,6。除了钙沉淀或物理技术,如电穿孔或显微注射使用专用装置7,现代化学方法已侧重于加强DNA细胞交付,同时降低细胞细胞毒性8, 9.使用脂质或阳离子聚合物形成脂质体状复合物,以及最近的非脂体聚合物化学系统使转染更容易和更有效率10。尽管有这些发展,细胞转染仍然需要具体技能,以准确执行,因为大多数物理或化学转染协议要求科学家手动准备每个DNA转染反应条件,因此影响吞吐量。为了规避这个问题,利用化学转染试剂11、12、13开发了反向转染方案,使用户能够更快地测试或组合几个质粒。在这些协议中,在将细胞播种到复合物上之前,与转染试剂形成核酸复合物。然而,这些反向协议仍然受到DNA溶液的手动处理和每个独立条件的组合的限制。虽然以96孔板格式进行它们是可行的,但DNA的制备和分配将单调乏味,并且可能会有错误。当需要不同数量的DNA质粒并相互多路复用时,细胞转染就更难实现,而且更加耗时,人为错误也变得相当不可避免。尽管很少多路复用DNA转染条件,但以反向转染方法扩展到384孔板格式,由于以下原因,成为一个不可能的挑战。i) 要管理的DNA量、转染试剂或反应混合物体积低于每口井的1μL。ii) 384个独立条件下质粒的多用性变得极其复杂。384口井的输送也非常耗时,而且容易出错。事实上,在预期井中分配正确的解决方案是很难管理的,因为已经分配的低容量不允许在空井和已满井之间进行目视监控。v) 最后,由于执行必要点胶步骤所需的时间,在加入细胞之前,蒸发干燥混合物的风险很高。总之,建立高通量DNA质粒转染测定的限制因素似乎是检测的小型化,这意味着小批量多路复用和管理,不能再手动处理,但也很难在由传统的围静态液体处理机的可靠方式。
为了证明自动化(如测定和获得高通量)的难度,到目前为止,只发布了几次自动转染的尝试:使用商业液体处理装置和磷酸钙沉淀的96 孔板格式最近,一个利波莱克斯试剂,和一个微流体芯片,使280个独立的转染15,但需要这方面的专业技能。另一种方法,去磷,允许液体悬浮,并导致流体操纵和混合,用于执行DNA转染在24至96孔板格式16。虽然可行,但这种方法的通量极低,因为细胞与DNA转染混合物的混合需要在播种前每一点孵育60s。这意味着整个 96 孔板的持续时间至少为 96 分钟。此外,由于这项工作是使用目前市场上没有的内部设计和制造设备完成的,因此该协议还远远不能让广大生物学家的观众满意。相反,在过去几年中,纳米体积分配器设备出现了一种易于使用的基于软件的声学点胶技术。利用聚焦声能,这些器件允许严格控制小液体体积从2.5 nL到500 nL从源板喷射到目的地17。这种技术称为声滴喷射 (ADE),具有许多优点:它是全自动的、非接触的、无提示的、准确、精确且高度可重复的,并且具有高通量18。首次致力于提供二甲基硫酸盐(DMSO)解决方案,设置已增强,以分配水缓冲液19。因此,声学纳米分配器似乎适用于反向细胞转染协议,并可以规避上述大多数手动限制。由于以前没有使用该技术描述过质粒转染尝试,我们最近评估了基于声学的点胶系统是否适合执行反向细胞转染。
利用纳米分给器的吞吐量和易用性,我们优化了HeLa细胞的反向转染方案,通过交叉测试几个参数,这些参数可以影响384孔单板的DNA转染,即总DNA量和源DNA起始浓度、稀释体积、转染试剂和扩散细胞数量。开发的协议规避了上述细胞转染的手动限制,与其他自动转染尝试具有若干优点。首先,它被小型化,从而允许通过节省DNA质粒制剂和转染试剂实现具有成本效益的转染试剂。其次,它比手动协议(即使是初学者)具有更高的吞吐量和可重现性,因为整个 384 孔板的转染可在 1 小时内实现。最后,它是软件驱动的,允许控制分配的DNA数量和多个质粒的多路复用。事实上,多亏了纳米分配器软件(材料表),用户可以制定一个研究计划来控制从定义的源井板到目的地的孔板的分配量。
此处提出的协议主要面向那些能够使用纳米分配器并希望在高通量下设置转染实验的用户,但也适用于那些希望通过以下形式快速优化特定细胞类型的转染参数的人。将该协议应用于在高吞吐量下交叉测试多个参数。事实上,我们已经表明,通过这种纳米尺度协议识别的优化参数可以转用于更大规模和手动转染实验。最后,由于本协议中使用的转染试剂允许根据制造商进行DNA或siRNA转染,该协议也对那些旨在执行基因过度表达或敲除的阵列方法的人感兴趣。预填充DNA的目的地板可在转染检测使用前7天保存,而不会失去功效,这是此类应用下协议的另一个优点。

<table border="0" cellpadding="0" cellspacing="0" style="width:1226px;" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384LDV Microplate</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;">LP-0200</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384-well Microplate &#956;Clear Black</td>
			<td style="width:363px;">Greiner</td>
			<td style="width:202px;">781906</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Ampicilin</td>
			<td style="width:363px;">Sigma</td>
			<td style="width:202px;">A9393-5G</td>
			<td style="width:291px;">Selection antibiotic for bacteria transformed with ampicilin expressing vector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Android Tablet</td>
			<td style="width:363px;">Samsung</td>
			<td style="width:202px;">Galaxy Note 8</td>
			<td style="width:291px;">used to guide the user while the source plate manual dispense</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Aniospray Surf 29</td>
			<td style="width:363px;">Anios</td>
			<td style="width:202px;">2421073</td>
			<td style="width:291px;">disinfectant to clean the MicroFlo head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Columbus software</td>
			<td style="width:363px;">Perkin Elmer</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">image analysis software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX Supplement, pyruvate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10566032</td>
			<td style="width:291px;">cell culture medium</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Echo Cherry Pick 1.5.3 software</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Software enabling ADE-based dispenses by the Echo550 device from a *.csv file; nanodispenser software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Echo550</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">ADE-based dispenser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Fetal Bovine Serum</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">16000044</td>
			<td style="width:291px;">to add in cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:363px;">Sigma-Aldrich</td>
			<td style="width:202px;">HT501128-4L</td>
			<td style="width:291px;">to fix cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HeLa cells</td>
			<td style="width:363px;">ATCC</td>
			<td style="width:202px;">HeLa (ATCC&#174; CCL-2&#8482;)</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">H3570</td>
			<td style="width:291px;">10 mg/mL Solution in Water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">INCell Analyzer 6000</td>
			<td style="width:363px;">GE Healthcare</td>
			<td style="width:202px;">29043323</td>
			<td style="width:291px;">automated laser-based confocal imaging platform</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:371px;">LB medium</td>
			<td style="width:363px;">Thermoischer Scientific 
			LB Broth Base (Lennox L Broth Base)&#174;, powder</td>
			<td style="width:202px;">12780052</td>
			<td style="width:291px;">culture medium for bacteria growth</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Lysis Buffer (A2)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740912.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">MicroFlo 10&#181;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170013</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">MicroFlo 1&#956;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170012</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">MicroFlo Dispenser</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7171000</td>
			<td style="width:291px;">peristaltic pump-based liquid handler device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microvolume spectrophotometer</td>
			<td style="width:363px;">Denovix</td>
			<td style="width:202px;">DS-11 Spectrophotometer</td>
			<td style="width:291px;">Measure the DNA concentration of samples</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:371px;">mVenus plasmid</td>
			<td style="width:363px;">mVenus cDNA was cloned by enzymatic restriction digestion and ligation in Age1/BsrG1 sites of the tdTomato-N1 plasmid</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: mVenus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Neutralization Buffer (A3)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740913.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">NucleoSpin Plasmid kit</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740588.50</td>
			<td style="width:291px;">used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Optimal-Modified Eagle Medium (Opti-MEM) Medium</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">31985070</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">optional Wash bufferWash Buffer (A4)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740914.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;">orbital shaker&#160;</td>
			<td style="width:363px;">incubated large capacity shaker</td>
			<td style="width:202px;">444-7084</td>
			<td style="width:291px;">Used to grow bacteria under gentle agitation and 37&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Penicillin-Streptomycin</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">15140122</td>
			<td style="width:291px;">10,000 U/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Phosphate-Buffered Saline</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10010001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Plasmid mini-columns</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740499.250</td>
			<td style="width:291px;">Silica membrane mini-column to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Resuspension Buffer (A1)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740911.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">RNAse A</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740505</td>
			<td style="width:291px;">Enzyme from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">tdTomato-N1 plasmid</td>
			<td style="width:363px;">Addgene</td>
			<td style="width:202px;">Plasmid #54642</td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: tdTomato&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">TransIT-X2 Dynamic Delivery System</td>
			<td style="width:363px;">Mirus Bio</td>
			<td style="width:202px;">MIR 6000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Wash Buffer (AW)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740916.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">3D printer</td>
			<td style="width:363px;">Creality</td>
			<td style="width:202px;">CR10S</td>
			<td style="width:291px;">used to print the plate adapter</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Blender Software&#160;</td>
			<td style="width:363px;">&#160;https://www.blender.org/ 
			Free software under GNU General Public License (GPL).</td>
			<td style="width:202px;">version 2.79b</td>
			<td style="width:291px;">used to design the plate adapter</td>
		</tr>
	</tbody>
</table>

high-throughput dna plasmid multiplexing and transfection using acoustic nanodispensing technology

细胞转染是许多生物学研究不可或缺的,需要控制许多参数,以实现准确和成功。通常在低吞吐量下执行,而且既耗时又容易出错,在多路复用多个质粒时更是如此。我们开发了一种简单、快速、准确的方法,利用声滴喷射 (ADE) 技术在 384 孔板布局中执行细胞转染。本研究中使用的纳米分配器装置基于该技术,允许从源井板高速向目标井板精确输送纳米体积。它可以根据预先设计的电子表格分配和复用DNA和转染试剂。在这里,我们提出了一个基于ADE的高通量质粒转染的最佳方案,这使得在共转染实验中达到高达90%的效率和近100%的共转染成为可能。我们扩展了初始工作,提出了一个基于用户友好的基于电子表格的宏,能够管理多达四个质粒/孔从包含多达1,536个不同的质粒的库,以及一个基于平板电脑的移液指南应用程序。宏设计了源板的必要模板,并为纳米分配器和基于平板电脑的应用生成即用型文件。四步转染协议涉及 i) 使用经典液体处理机的稀释剂,ii) 质粒分布和多路复用,iii) 纳米分配器的转染试剂点,以及 iv) 预填充孔上的细胞电镀。所述基于软件的ADE质粒多路复用和转染控制,即使是该领域的非专家也能快速、安全地执行可靠的细胞转染。此方法能够快速识别给定单元类型的最佳设置,并可转用到更大规模和手动的方法。该协议简化了非池化筛选策略中的人类ORFeome蛋白(基因组中开放阅读框架[ORF])表达或基于CRISPR-Cas9的基因功能验证等应用。

f 为了确定ADE技术是否可用于自动反向转染协议,我们使用红色荧光tdTomato表达质粒,通过荧光显微镜监测细胞转染效率。首先,为了确定最佳转染参数,对不同的稀释量和总DNA进行了交叉测试。稀释体积用于允许DNA液滴,一旦分配,蔓延到整个井,以规避在初步实验中观察到的不均匀转染(即,仅在井的中心)。如图6A所示,使用脂聚聚物试剂20转...

Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

该协议使用声滴喷射技术描述了384孔板中哺乳动物细胞的高通量质粒转染。耗时、易出错的 DNA 点胶和复用,以及转染试剂点胶,由软件驱动,由纳米点胶设备执行。然后,这些细胞被播种在这些预填充的井中。

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

本视频中开发的基于 Nano 组件的转染协议代表了第一种用户友好、高通量转染方法，甚至允许该领域的初学者取得成功。这在高通量技术上很容易，允许具有 384 独立条件的细胞转染。到目前为止，由于需要纳米体积的试剂，该方法成本低。为了定义所有细胞类型的最佳转染参数，我们建议保持源DNA浓度恒定，并使用不同数量的转染DNA、转染试剂和细胞数量。要分配 40 微升电池悬浮液，请将 10 微升盒式磁带安装在近光液体处理设备上，并准备适当的程序。首先，将流速参数调整为低，以低速度分配细胞，以避免通过纯粹的应力和高冲击井底对细胞造成潜在损害。然后，将分配高度调整为 11.43 毫米。在点胶过程中，高度必须足够高，以降低孔底的电池冲击，但足够低，以避免在点胶头上保留液滴。接下来，将板透明高度调整为 16 毫米，以便点胶每排后将点胶头自由位移在板上。目视控制近视液体处理者头部高度的正确设置。在点胶时，确保点胶尖端上没有掉落。还要验证头部是否足够高，以允许在分配每一行后对头部进行位移。为了生成选择列表来驱动 ADE 分配，开发了一个用户友好的电子表格宏，用于管理 DNA 量，并在 384 井板格式中混合多达四个质粒。最佳卷在电子表格的某些单元格中预先填充，但可以更改。在粉红色字段中，转染试剂（或 TR 混合物值）设置为 500 纳米光。源板井中的最小体积值设置为四微升，源板井的最大体积设置为 11.25 微升。输入100纳米每个微升DNA开始浓度在蓝色领域对应的基础DNA。然后，在与板的 384 孔对应的灰色和绿色字段中输入所需的 DNA 量。输入数量和质粒名称，并确保如果相同的质粒必须在几个孔中转染，请使用相同的拼写。单击生成选取列表以验证输入的值，如果请求，更正橙色填充单元格，指示错误或卷无法由 Nano 组件处理。如果未检测到任何情况，宏将从相应图纸上收集的数据生成 384 井点胶指南、DNA 选取列表文件和 TR 选取列表文件。从源板打印模板，以可视化在点胶前要填充的油井。指示质粒名称和最小填充量。转染试剂混合物体积，将填充在以下孔，指示为TR，并突出显示为绿色。要准备DNA源板，使用蒸馏水将储存的DNA质粒稀释至每微升100纳米。现在，将 384 井网格校准到板尺寸。在平板电脑上打开 384 井移液指南应用。将源位置放在下部屏幕上的网格上。在左上角校准菜单中，单击正负可增强或减小网格和井的大小，以便将绿色井调整到板的四角井。使用双面胶带，将 3D 打印板适配器安装在屏幕上，以避免在点胶时发生源板移动。如果需要，使用旋转箭头和向上、向下、向右、向左按钮移动已校准的网格，以将网格调整到板位置。一旦正确校准和定位网格和井尺寸，勾选锁校准框。单击文件并打开 384-井-管道指南。csv 文件。按照屏幕说明手动将指定浓度的指定浓度的质粒指示体积分配到与预期板的正确目标相对应的白色高亮井中。在DNA点胶过程中，使用减或加箭头返回或进一步。到达第一个转染试剂溶液进行加载时停止点胶。DNA 分配完成后，从适配器上取出源板。如果要填充多个源板，请将新的源板放在适配器上，然后按照点胶说明进行。离心DNA填充源板，以确保适当的液体调平，并消除气泡，导致在 ADE 基转移不准确。现在，运行一个调查来控制手动分配的卷。在纳米组件 PC 上，运行纳米组件程序。转到诊断选项卡，勾选源板出箱，将源板加载到板架上，然后勾选进入板。当提示时，384LDV_AQ_B2将 Nano 分配器设置为水缓冲缓冲器点胶模式，然后按"确定"。在杂项菜单中选择调查。然后点击启动。选择要分析并单击"转到"按钮的预填充井。验证测量的体积与预期体积匹配，并确保未装载超过 12 微升的孔。用无血清介质填充新的无菌容器，用10毫升预热无血清介质填充管子，并淹没管内组织者。按近位液体处理器的底按钮约 10 秒钟。通过目视检查点头的流量，确保尖端没有堵塞。将无菌 384 井培养板放在近位液体处理板托架上，并取下其盖子。运行预校准程序，在 384 井板的每个井中分配一个微升。点胶时间约为 8 秒。然后更换 384 井板的盖子。要设置 ADE 驱动的 DNA 点胶，请运行选取列表软件。将源的 384 孔设置为 384LDV。通过选择"水缓冲器"将设备设置为水缓冲384LDV_AQ_B2。和目标板类型Greiner_384PS_781096。取消勾选优化传输吞吐量。选择选取列表选项卡。单击导入。并选择DNA_Picklist_CSV文件。然后，单击播放并保存协议。单击模拟以执行程序分配的模拟，以确保选取列表与预期的实验设计匹配。单击运行按钮并启动点胶程序。当询问时，插入请求的源板。然后在纳米分配器中的目的地板。要建立转染试剂的分离，在生物安全柜中工作，将无血清介质中的脂质复性转染试剂稀释至1 x最终浓度，并涡旋管。立即根据宏的预定义源板，并使用预校准的 384 井移液导轨应用分配 TR 混合。TR 点胶后，执行一项调查以控制 TR 加载的体积，就像以前对加载的 DNA 所做的一样。选择要分析的转染反应混合预填充井。然后点击"转到"按钮。验证测量的体积与预期的体积匹配。并确保没有井装了超过12微升的体积。现在，在选取列表软件中执行 DNA 选择列表的重置。验证设备参数是否仍设置为水缓冲区。输入正确的源和目标板类型。在"选取列表"选项卡上，单击重置以清除示例列表。然后单击导入并选择TR_Picklist。CSV 文件。点击播放。如果提示并执行程序转染试剂混合物分配的模拟，请保存协议，通过单击模拟按钮确保正确的设计。如前所述，单击运行按钮并启动点胶程序后，将源板和目标板按要求放在 NanoDipsenser 中。要分配细胞，请用准备好的细胞悬浮液填充新的无菌容器并搅拌它，以避免沉积导致不准确和细胞密度。在溶液中插入管管理器。然后，按下底按钮，直到电池悬架开始从点胶头冲洗。通过在冲洗时目视检查来自点胶头的流量，确保尖端没有堵塞，并确保每个管都装有电池悬架。现在，将DNA和TR填充的384井目标板加载到近源式液体处理板载体上，并取下其盖子。运行预校准程序，在完整的 384 井板上分配 40 微升的电池悬架。点胶时间约为 45 秒。最后，更换384井板的盖子。利用脂复试剂对希拉细胞的转染成功。从5到30纳米的DNA量在一个微升稀释剂体积上显示出相同的效率，高达90%的细胞转染。相比之下，较高的量导致转染细胞的百分比突然下降。测试了从15纳米升到4个微升的各种稀释剂体积，并确定了1个微升的最佳条件。为了进一步提高该协议的吞吐量，我们测试了两种有效的DNA存储方法，即干存储板或冷冻存储。这两种存储方法没有导致与新分配的 DNA 溶液存储长达七天的结果显著不同。由于血浆转染通常至少使用两种不同的质粒，因此使用最佳识别条件对该协议的DNA多路复用能力进行了检查。先前使用的tdTomato红色荧光蛋白表达质粒被修改，以表达mVenus一种明亮的黄色荧光蛋白，然后两者都用于共转染尝试。红色或绿色荧光阳性细胞分析表明，转染效率约为80%，然而，近100%的红细胞还与mVenus表达质粒的mVenus进行共膜移植，这可以从基于软件的代表性图像分析中看出。一旦DNA在源板中分配。执行调查以确保已加载预期卷且不超过 12 微升。在源板中分配转染试剂时，尽量避免气泡，因为离心子板将导致转染功效完全丧失。该方法可应用于生物实验、犯罪和转染，适用于大部分可采用384井板格式进行。全防转染为新应用铺平了道路，例如以脆铸非筛选方法为名，这种质粒已知采用非筛选方法，能够监测间歇性影响，目前无法以不良策略进行排序。

Research

JoVE Journal

Genetics

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...