Protein and mRNA Quantification in Small Samples of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 96-Well Microplates.
Methods Mol Biol
; 2485: 15-37, 2022.
Article
em En
| MEDLINE
| ID: mdl-35618896
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células-Tronco Pluripotentes Induzidas
Limite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Estados Unidos