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High-throughput RNAi screening in vitro: From cell lines to primary cells
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href="/search?author1=DMITRIY+OVCHARENKO&sortspec=date&submit=Submit">DMITRIY OVCHARENKO</a></span><a class="xref-" href="#aff-1" id="xref-aff-1-1">1</a><span class="xref-sep">,</span><a class="xref-" href="#aff-2" id="xref-aff-2-1">2</a>, </li> <li id="contrib-2"><span class="name"><a class="name-search" href="/search?author1=RICHARD+JARVIS&sortspec=date&submit=Submit">RICHARD JARVIS</a></span><a class="xref-" href="#aff-1" id="xref-aff-1-2">1</a>, </li> <li id="contrib-3"><span class="name"><a class="name-search" href="/search?author1=SCOTT+HUNICKE-SMITH&sortspec=date&submit=Submit">SCOTT HUNICKE-SMITH</a></span><a class="xref-" href="#aff-1" id="xref-aff-1-3">1</a>, </li> <li id="contrib-4"><span class="name"><a class="name-search" href="/search?author1=KEVIN+KELNAR&sortspec=date&submit=Submit">KEVIN KELNAR</a></span><a class="xref-" href="#aff-1" id="xref-aff-1-4">1</a>, and </li> <li id="contrib-5"><span class="name"><a class="name-search" href="/search?author1=DAVID+BROWN&sortspec=date&submit=Submit">DAVID BROWN</a></span><a class="xref-" href="#aff-1" id="xref-aff-1-5">1</a></li> </ol> <ol class="affiliation-list"> <li class="aff"><a id="aff-1" name="aff-1"></a><address><sup>1</sup>Ambion, Inc., Austin, Texas 78744, USA </address> </li> <li class="aff"><a id="aff-2" name="aff-2"></a><address><sup>2</sup>Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, USA </address> </li> </ol> </div> <div class="section abstract" id="abstract-1" itemprop="description"> <h2>Abstract</h2> <p id="p-1">Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale. </p> <h3 class="kwd-header">Keywords</h3> <ul class="kwd-group"> <li class="kwd"><span><a class="kwd-search" href="/search?fulltext=RNAi%20screening&sortspec=date&submit=Submit&andorexactfulltext=phrase">RNAi screening</a></span></li> <li class="kwd"><span><a class="kwd-search" href="/search?fulltext=gene%20expression&sortspec=date&submit=Submit&andorexactfulltext=phrase">gene expression</a></span></li> <li class="kwd"><span><a class="kwd-search" href="/search?fulltext=siRNA&sortspec=date&submit=Submit&andorexactfulltext=phrase">siRNA</a></span></li> <li class="kwd"><span><a class="kwd-search" href="/search?fulltext=reverse%20transfection&sortspec=date&submit=Submit&andorexactfulltext=phrase">reverse transfection</a></span></li> <li class="kwd"><span><a class="kwd-search" href="/search?fulltext=electroporation&sortspec=date&submit=Submit&andorexactfulltext=phrase">electroporation</a></span></li> </ul> </div> <p id="p-2"></p> <div class="section fn-group" id="fn-group-1"> <h2>Footnotes</h2> <ul> <li class="fn" id="fn-1"> <p id="p-64">Article and publication are at <a href="http://www.rnajournal.org/cgi/doi/10.1261/rna.7288405">http://www.rnajournal.org/cgi/doi/10.1261/rna.7288405</a>. </p> </li> <li class="history" id="history-1"> <ul class="history-list"> <li xmlns:hwp="http://schema.highwire.org/Journal" class="accepted" hwp:start="2005-02-26">Accepted February 26, 2005.</li> <li xmlns:hwp="http://schema.highwire.org/Journal" class="received" hwp:start="2004-12-28">Received December 28, 2004.</li> </ul> </li> <li class="copyright-statement" id="copyright-statement-1">Copyright 2005 by RNA Society</li> </ul> </div><span class="highwire-journal-article-marker-end"></span></div> <div class="social-bookmarking"> <ul 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