Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells. A single-protein counting method called digital PLA was developed, allowing ultrasensitive measurement of absolute protein and mRNA copy numbers from single mammalian cells. Joint single-cell mRNA/protein measurements were then used to construct and validate a stochastic two-state model for transcription and translation of the gene CD147.
Digital Quantification of Proteins and mRNA in Single Mammalian Cells / Albayrak, C.; Jordi, C. A.; Zechner, C.; Lin, J.; Bichsel, C. A.; Khammash, M.; Tay, S.. - In: MOLECULAR CELL. - ISSN 1097-2765. - 61:6(2016), pp. 914-924. [10.1016/j.molcel.2016.02.030]
Digital Quantification of Proteins and mRNA in Single Mammalian Cells
Zechner C.;
2016-01-01
Abstract
Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells. A single-protein counting method called digital PLA was developed, allowing ultrasensitive measurement of absolute protein and mRNA copy numbers from single mammalian cells. Joint single-cell mRNA/protein measurements were then used to construct and validate a stochastic two-state model for transcription and translation of the gene CD147.File | Dimensione | Formato | |
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