Version 1
: Received: 10 December 2021 / Approved: 13 December 2021 / Online: 13 December 2021 (16:13:27 CET)
How to cite:
Li, W.; Han, J.; Entcheva, E. Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates. Preprints2021, 2021120215. https://doi.org/10.20944/preprints202112.0215.v1
Li, W.; Han, J.; Entcheva, E. Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates. Preprints 2021, 2021120215. https://doi.org/10.20944/preprints202112.0215.v1
Li, W.; Han, J.; Entcheva, E. Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates. Preprints2021, 2021120215. https://doi.org/10.20944/preprints202112.0215.v1
APA Style
Li, W., Han, J., & Entcheva, E. (2021). Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates. Preprints. https://doi.org/10.20944/preprints202112.0215.v1
Chicago/Turabian Style
Li, W., Julie Han and Emilia Entcheva. 2021 "Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates" Preprints. https://doi.org/10.20944/preprints202112.0215.v1
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 WesTM by ProteinSimple and the Power SYBRTM Green Cells-to-CTTM 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.
Medicine and Pharmacology, Cardiac and Cardiovascular Systems
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
