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Identification of valid reference housekeeping genes for gene expression analysis in tumor neovascularization studies

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Abstract

Introduction

Real time RT-PCR is a widely used technique to evaluate and confirm gene expression data obtained in different cell systems and experimental conditions. However, there are many conflicting reports about the same gene or sets of gene expression. A common method is to report the interest gene expression relative to an internal control, usually a housekeeping gene (HKG), which should be constant in cells independently of experimental conditions.

Materials and Methods

In this study, the expression stability of ten HKGs was considered in parallel in two cell systems (endothelial and osteosarcoma cells): beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Cyclophilin A (PPIA), beta-2-microglobulin (B2M), glucuronidase beta (GUSB), eukaryotic translation elongation factor 1 alpha1 (EEF1A1), transferrin receptor (TFRC), ribosomal protein S18 (RPS18). In order to study the stability of candidate reference genes, data have been also analyzed by several algorithms (geNorm, NormFinder, BestKeeper and delta-Ct method).

Results and Conclusions

The overall analysis obtained by the comprehensive ranking showed that RPS18 and PPIA are appropriate internal reference genes for tumor neovascularization studies where it is necessary to analyze both systems at the same time.

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Conflict of interest

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Authors and Affiliations

  1. Department of General Pathology, Second University of Naples, Via L. De Crecchio 7, 80138, Naples, Italy

    Monica Rienzo, Concetta Schiano & Amelia Casamassimi

  2. Department of General Pathology, Chair of Clinical Pathology, Excellence Research Centre on Cardiovascular Disease, U.O.C. Immunohematology, Transfusion Medicine and Transplant Immunology (SIMT), Regional Reference Laboratory of Transplant Immunology (LIT), Azienda Universitaria Policlinico (AOU), 1st School of Medicine, Second University of Naples, Piazza Miraglia 2, 80138, Naples, Italy

    Vincenzo Grimaldi & Claudio Napoli

  3. IRCCS SDN Foundation, Via Gianturco 113, 80134, Naples, Italy

    Teresa Infante & Claudio Napoli

Authors
  1. Monica Rienzo
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  2. Concetta Schiano
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  3. Amelia Casamassimi
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  4. Vincenzo Grimaldi
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  5. Teresa Infante
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  6. Claudio Napoli
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Corresponding author

Correspondence to Monica Rienzo.

Additional information

M. Rienzo and C. Schiano contributed equally to this study.

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Rienzo, M., Schiano, C., Casamassimi, A. et al. Identification of valid reference housekeeping genes for gene expression analysis in tumor neovascularization studies. Clin Transl Oncol 15, 211–218 (2013). https://doi.org/10.1007/s12094-012-0904-1

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  • DOI: https://doi.org/10.1007/s12094-012-0904-1

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