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Nat Methods. 2015 Aug;12(8):739-42. doi: 10.1038/nmeth.3446. Epub 2015 Jun 22.

Combining protein and mRNA quantification to decipher transcriptional regulation.

Author information

1
1] Verna &Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA. [2] Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [3] Center for Theoretical Biological Physics, Rice University, Houston, Texas, USA.
2
1] Verna &Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA. [2] Center for Theoretical Biological Physics, Rice University, Houston, Texas, USA.
3
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas, USA.
4
Verna &Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.

Abstract

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.

PMID:
26098021
PMCID:
PMC4521975
DOI:
10.1038/nmeth.3446
[Indexed for MEDLINE]
Free PMC Article

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