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Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR

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Abstract

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan-endothelial cell markers murine CD31 and vascular endothelial-cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or β-actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non-endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2.

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Acknowledgments

This work was supported in part by grants from Ministero dell’Istruzione, Università e Ricerca (MIUR, Centro IDET, FIRB project RBAP11H2R9 2011) and Associazione Italiana per la Ricerca sul Cancro (AIRC grant no 10396) to MP.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Chiara Testini

    Present address: Department of Immunology, Genetics and Pathology-IGP, Uppsala University, Uppsala, Sweden

Authors and Affiliations

  1. Unit of Histology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy

    Daniela Coltrini

  2. Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa 11, 25123, Brescia, Italy

    Emanuela Di Salle, Roberto Ronca, Mirella Belleri, Chiara Testini & Marco Presta

Authors
  1. Daniela Coltrini
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  2. Emanuela Di Salle
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  3. Roberto Ronca
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  4. Mirella Belleri
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  5. Chiara Testini
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  6. Marco Presta
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Corresponding author

Correspondence to Marco Presta.

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Coltrini, D., Di Salle, E., Ronca, R. et al. Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR. Angiogenesis 16, 469–477 (2013). https://doi.org/10.1007/s10456-012-9324-7

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  • DOI: https://doi.org/10.1007/s10456-012-9324-7

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