Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China.
PLoS One. 2012;7(6):e39419. doi: 10.1371/journal.pone.0039419. Epub 2012 Jun 27.
The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.
化学合成的 siRNA 双链体已成为 RNAi 功能丧失研究的强大且广泛应用的工具，但存在高脱靶效应问题。最近，内切核酸酶制备的 siRNA（esiRNA）由于其较低的脱靶效应和成本效益而被证明是一种有吸引力的替代方法。然而，esiRNA 的当前制造方法很复杂，主要是在大规模水平上的纯化和归一化。在这项研究中，我们提出了一种磁珠集成芯片，该芯片可以将扩增或转录产物固定在磁珠上，并以稳健且方便的方式完成转录、消化、归一化和纯化。该芯片能够在大规模水平上制造即用型 esiRNA。在转录、翻译和功能水平上验证了这些 esiRNA 的沉默特异性和效率。在单个孔中成功制造了几种归一化的 esiRNA，包括沉默 PARP1 和 BRCA1 的 esiRNA，并随后利用它们有效地研究它们对细胞活力的协同作用。构建了一个针对 68 个酪氨酸激酶基因的小 esiRNA 文库进行功能丧失研究，鉴定出四个基因调节 Hela 细胞的迁移能力。我们相信，与当前方法相比，这种方法为制造 esiRNA 提供了更强大且更具成本效益的选择，因此这些异质 RNA 链可能在大多数密集和广泛的应用中具有实用性。
