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Analysis of Suppressor of Cytokine Signalling (SOCS) Gene Expression by Real-Time Quantitative PCR

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 967)

Abstract

The Suppressor of Cytokine Signalling (SOCS) proteins are a family of negative regulators characterized by a central SH2 domain and C-terminal SOCS box motif. Cytokine Inducible SH2-containing protein (CIS), SOCS1, 2 and 3 are rapidly upregulated in response to cytokine stimulation and act to inhibit JAK/STAT signalling by a variety of mechanisms. The expression of SOCS proteins provides a level of specificity in the control of signalling, with SOCS proteins differentially upregulated in response to individual cytokines and in various cell-types. Real-time reverse transcription (RT) quantitative polymerase chain reaction (RT-qPCR) is an established technique for quantifying mRNA in biological samples, measuring the relative expression of genes of interest and identifying single nucleotide polymorphisms. Here we describe the use of SYBR® Green I RT-qPCR to quantify the relative expression level of SOCS mRNA in murine bone marrow-derived macrophages (BMDM). The approach can be universally applied to different cell types and various tissues.

Key words

RT-quantitative PCR SOCS Suppressor of cytokine signalling Macrophages Reference gene Standard curve Ct value Relative expression 
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Notes

Acknowledgments

This research was supported by the NHMRC Australia (Program Grant 461219 and fellowship to S.E.N.) and an NIH Grant (CA022556). It was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Inflammation DivisionWalter and Eliza Hall Institute of Medical ResearchParkvilleAustralia
  2. 2.University of MelbourneParkvilleAustralia

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