title: Digital Quantification of Proteins and mRNA in Single Mammalian Cells.
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.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Cem
authors.initials: C
authors.lastname: Albayrak
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Christian A
authors.initials: CA
authors.lastname: Jordi
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Zechner
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Jing
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Colette A
authors.initials: CA
authors.lastname: Bichsel
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Khammash
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland; Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA. Electronic address: tays@uchicago.edu.
authors.forename: Savaş
authors.initials: S
authors.lastname: Tay
mesh_terms: D000818:Animals; D051926:Basigin / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D051926:Basigin