Digital Quantification of Proteins and mRNA in Single Mammalian Cells

被引：131

作者：

Albayrak, Cem ^{[1
]}

Jordi, Christian A. ^{[1
]}

Zechner, Christoph ^{[1
]}

Lin, Jing ^{[1
]}

Bichsel, Colette A. ^{[1
,3
]}

Khammash, Mustafa ^{[1
]}

Tay, Savas ^{[1
,2
]}

机构：

[1] ETH, Dept Biosyst Sci & Engn, CH-4058 Basel, Switzerland

[2] Univ Chicago, Inst Mol Engn, Chicago, IL 60637 USA

[3] Univ Bern, Ctr Biomed Engn, CH-3010 Bern, Switzerland

来源：

MOLECULAR CELL | 2016年 / 61卷 / 06期

关键词：

DNA COPY NUMBER; STOCHASTIC GENE-EXPRESSION; PROXIMITY LIGATION; QUANTITATIVE PCR; SIGNALING PATHWAYS; VARIABILITY; CYTOMETRY; ASSAYS; NOISE; CHIP;

D O I：

10.1016/j.molcel.2016.02.030

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：914 / 924

页数：11

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