Department of Biomedical Engineering, Peking University, Yi He Yuan Road No. 5, China.
Lab Chip. 2011 Mar 21;11(6):1036-40. doi: 10.1039/c0lc00564a. Epub 2011 Jan 28.
Endoribonuclease-prepared siRNAs (esiRNAs) have the advantages of cost effectiveness and lower off-target effects than chemically synthesized siRNA. However, the current manufacture of esiRNA is a complex process, requiring an expensive instrument and demanding skills to accomplish the transfer, purification, quantification and normalization of liquid samples. These performances significantly hamper the application of esiRNAs on a large-scale level. In this study, we present a polymer microbead-integrated chip capable of the large-scale manufacture of esiRNA in a convenient and robust manner. This chip is able to perform the amplification, transcription and enzymatic digestion of targets on polymer scaffold, thus simplifying the transfer and purification manipulation process. What is also noted, this chip can readily tailor and normalize the amount of esiRNA product by controlling the number of DNA probes and the cycle of the amplification reaction. Thus the esiRNA, also referred to as gel-esiRNA, can be immediately applied to loss-of-function study without any further treatment. The silencing specificity and efficiency of gel-esiRNAs were assessed on transcriptional, translational or cell functional levels. All data of real-time PCR, Western blot assay, or FACS clearly supported that the gel-esiRNA produced specific gene silencing as effectively as the one generated following the conventional approach. We believe that this approach would provide a more robust and cost-effective choice to manufacture esiRNAs, thus promising both more intensive and extensive applications of these heterogeneous RNA strands.
内切酶制备的 siRNA（esiRNA）具有成本效益高和脱靶效应低的优点，优于化学合成的 siRNA。然而，目前 esiRNA 的制造是一个复杂的过程，需要昂贵的仪器和技能来完成液体样品的转移、纯化、定量和标准化。这些性能极大地阻碍了 esiRNA 在大规模水平上的应用。在本研究中，我们提出了一种聚合物微珠集成芯片，能够以方便和稳健的方式大规模制造 esiRNA。该芯片能够在聚合物支架上进行目标的扩增、转录和酶消化，从而简化了转移和纯化操作过程。值得注意的是，该芯片可以通过控制 DNA 探针的数量和扩增反应的循环来轻松调整和标准化 esiRNA 产物的数量。因此，这种 esiRNA 也称为凝胶 esiRNA，可以立即应用于功能丧失研究，而无需进一步处理。我们在转录、翻译或细胞功能水平上评估了凝胶 esiRNA 的沉默特异性和效率。实时 PCR、Western blot 分析或 FACS 的所有数据都清楚地表明，凝胶 esiRNA 能够像传统方法一样有效地产生特定的基因沉默。我们相信，这种方法将为制造 esiRNA 提供更强大和更具成本效益的选择，从而有望更深入和广泛地应用这些异质 RNA 链。
