Protein and mRNA Quantification in Small Samples of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 96-Well Microplates.
Protein and mRNA Quantification in Small Samples of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 96-Well Microplates.
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DOI：
10.1007/978-1-0716-2261-2_2
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发表时间：
2022
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我们描述了一种方法，用于蛋白质定量和mRNA定量的小样本量的人诱导的多能干细胞衍生的心肌细胞（hiPSC-CM）。本文展示了如何将ProteinSimple的基于毛细管的蛋白质检测系统Wes™和Invitrogen的Power SYBR™绿色Cells-to-CT™试剂盒应用于96孔微孔板格式的单个样品，从而与高通量（HT）心肌细胞测定兼容。作为使用的一个例子，我们说明了当光遗传学致动器通道视紫红质-2（ChR 2）在hiPSC-CM中遗传表达时，hiPSC-CM中的Cx43蛋白和GJA 1 mRNA水平增强。提供了hiPSC-CM细胞培养和裂解物制备的说明，沿着优化的参数设置和实验方案步骤。讨论了优化一抗浓度的策略以及信号归一化的方法，即，抗体多重化和总蛋白测定。Wes和Cells-to-CT试剂盒的灵敏度使HT格式的蛋白质和mRNA定量成为可能，这在处理珍贵的小样本时非常重要。除了能够处理小的心肌细胞样品外，这些简化和半自动化的过程还可以进行快速的机械分析。
We describe a method for protein quantification and for mRNA quantification in small sample quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system Wes™ by ProteinSimple and the Power SYBR™ Green Cells-to-CT™ Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e., antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.