Laboratory of Chronobiology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Institute for Theoretical Biology, Humboldt-Universität zu Berlin, Berlin, Germany.
Methods Mol Biol. 2021;2130:103-114. doi: 10.1007/978-1-0716-0381-9_8.
RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.
RNA 干扰 (RNAi) 通过中和靶向 mRNA 分子来实现基因表达的选择性下调，已广泛应用于高通量筛选研究中。在这里，我们描述了一种用于高效平行 RNAi 介导的基因表达下调的方案，以寻找参与生物钟生成的成分。我们使用慢病毒基因转移将 shRNA 表达质粒递送至生物钟报告细胞，以确保高效稳定的敲低。使用同步报告细胞的活细胞生物发光记录来监测生物钟节律，可在数天内进行。此外，我们还介绍了一种新的软件工具（ChronoStar），用于高效、平行的时间序列分析，以提取节律参数，如周期、相位、幅度和衰减。
