Unique reporter-based sensor platforms to monitor signalling in cells

Meesbah Jiwaji, Rónán Daly, Abdullah Gibriel, Gráinne Barkess, Pauline McLean, Jingli Yang, Kshama Pansare, Sarah A. Cumming, Alisha McLauchlan, Piotr J. Kamola, Musab S. Bhutta, Adam G. West, Katherine L. West, Walter Kolch, Mark A. Girolami, Andrew R. Pitt

Research output: Contribution to journalArticlepeer-review

Abstract

Introduction - In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level.
Methods - Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis.
Findings - We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation.
Conclusions - We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.
Original languageEnglish
Article numbere50521
Number of pages10
JournalPLoS ONE
Volume7
Issue number11
DOIs
Publication statusPublished - 29 Nov 2012

Bibliographical note

© 2012 Jiwaji et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords

  • real-time polymerase chain reaction
  • dexamethasone
  • Western blotting
  • transcription factors
  • oligonucleotide array sequence analysis
  • humans
  • systems biology
  • plasmids
  • cell line
  • cadmium chloride
  • forskolin

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