Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR

Joeri L. Aerts, Monica I. Gonzales, Suzanne L. Topalian

Research output: Contribution to journalArticlepeer-review


Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for β-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Original languageEnglish (US)
Pages (from-to)84-91
Number of pages8
Issue number1
StatePublished - Jan 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)


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