Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology(KIST), Seoul, KS013, Korea.
Department of Chemical Biological Engineering, Korea University, Seoul, KS013, Korea.
Sci Rep. 2021 Mar 19;11(1):6463. doi: 10.1038/s41598-021-85728-y.
Given the growing interest in molecular diagnosis, highly extensive and selective detection of genetic targets from a very limited amount of samples is in high demand. We demonstrated the highly sensitive and multiplexed one-step RT-qPCR platform for RNA analysis using microparticles as individual reactors. Those particles are equipped with a controlled release system of thermo-responsive materials, and are able to capture RNA targets inside. The particle-based assay can successfully quantify multiple target RNAs from only 200 pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. We carried out 8-plex one-step RT-qPCR using tens of microparticles, which allowed extensive mRNA profiling. The circadian cycles were shown by the multiplex one-step RT-qPCR in human cell and human hair follicles. Reliable 24-plex one-step RT-qPCR was developed using a single operation in a PCR chip without any loss of performance (i.e., selectivity and sensitivity), even from a single hair. Many other disease-related transcripts can be monitored using this versatile platform. It can also be used non-invasively for samples obtained in clinics.
鉴于人们对分子诊断越来越感兴趣，因此非常需要从非常有限的样本量中高度广泛和选择性地检测遗传靶标。我们展示了一种使用微珠作为单个反应器的高度敏感和多重一步 RT-qPCR 平台，用于 RNA 分析。这些颗粒配备了热响应材料的控制释放系统，能够捕获内部的 RNA 靶标。基于颗粒的测定法能够成功地从仅 200 pg 的总 RNA 中定量多种靶标 RNA。该测定法还可以借助预浓缩过程从单个细胞中定量靶标 RNA。我们使用数十个微珠进行了 8 重一步 RT-qPCR，从而可以广泛进行 mRNA 分析。在人类细胞和人类毛囊中，通过多重一步 RT-qPCR 显示了昼夜节律循环。即使仅从一根头发中，也可以通过单个操作在 PCR 芯片中进行可靠的 24 重一步 RT-qPCR，而不会降低性能（即选择性和灵敏度）。使用这种多功能平台还可以监测许多其他与疾病相关的转录本。它还可以用于从临床获得的非侵入性样本。
