Department of Precision Mechanics, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan.
Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, Japan.
Sci Rep. 2018 Jun 15;8(1):9214. doi: 10.1038/s41598-018-27547-2.
We assessed the applicability of giant unilamellar vesicles (GUVs) for RNA detection using in vesicle reverse transcription polymerase chain reaction (RT-PCR). We prepared GUVs that encapsulated one-pot RT-PCR reaction mixture including template RNA, primers, and Taqman probe, using water-in-oil emulsion transfer method. After thermal cycling, we analysed the GUVs that exhibited intense fluorescence signals, which represented the cDNA amplification. The detailed analysis of flow cytometry data demonstrated that rRNA and mRNA in the total RNA can be amplified from 10-100 copies in the GUVs with 5-10 μm diameter, although the fraction of reactable GUV was approximately 60% at most. Moreover, we report that the target RNA, which was directly transferred into the GUV reactors via membrane fusion, can be amplified and detected using in vesicle RT-PCR. These results suggest that the GUVs can be used as biomimetic reactors capable of performing PCR and RT-PCR, which are important in analytical and diagnostic applications with additional functions.
我们评估了使用囊泡内反转录聚合酶链反应（RT-PCR）检测 RNA 的巨大单层囊泡（GUVs）的适用性。我们使用油包水乳剂转移法制备了包含模板 RNA、引物和 Taqman 探针的一锅 RT-PCR 反应混合物的 GUVs。在热循环后，我们分析了表现出强烈荧光信号的 GUVs，这代表了 cDNA 扩增。通过对流式细胞术数据的详细分析，我们表明可以从直径为 5-10 μm 的 GUV 中扩增出总 RNA 中的 rRNA 和 mRNA，其拷贝数为 10-100，尽管最多只有大约 60%的 GUV 是可反应的。此外，我们报告称，通过膜融合直接转移到 GUV 反应器中的靶 RNA 可以使用囊泡内 RT-PCR 进行扩增和检测。这些结果表明，GUV 可用作具有额外功能的在分析和诊断应用中进行 PCR 和 RT-PCR 的仿生反应器。
