Original ArticlesOptimal Reference Genes for Normalization of qRT-PCR Data from Archival Formalin-fixed, Paraffin-embedded Breast Tumors Controlling for Tumor Cell Content and Decay of mRNATramm, Trine MD; Sørensen, Brita S. PhD; Overgaard, Jens MD, DMSc; Alsner, Jan PhD Author Information Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus C, Denmark Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.molecularpathology.com. The authors declare no conflict of interest. Reprints: Trine Tramm, MD, Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, Building 5, DK-8000 Aarhus C, Denmark (e-mail: [email protected]). Diagnostic Molecular Pathology 22(3):p 181-187, September 2013. | DOI: 10.1097/PDM.0b013e318285651e Buy SDC Metrics Abstract Reliable determination of gene-expression levels from qRT-PCR requires accurate normalization of target genes to reference genes in order to remove nonbiological variation. Reference genes are ideally constitutively expressed in every cell, but many genes used for normalization has been shown to vary with tissue type, cellular proliferation, cancer progression, and degradation of nucleic acids. Gene-expression analysis is increasingly performed on degraded mRNA from formalin-fixed, paraffin-embedded tissue (FFPE), giving the option of examining retrospective cohorts. The aim of this study was to select robust reference genes showing stable expression over time in FFPE, controlling for various content of tumor tissue and decay of mRNA because of variable length of storage of the tissue. Sixteen reference genes were quantified by qRT-PCR in 40 FFPE breast tumor samples, stored for 1 to 29 years. Samples included 2 benign lesions and 38 carcinomas with varying tumor content. Stability of the reference genes were determined by the geNorm algorithm. mRNA was successfully extracted from all samples, and the 16 genes quantified in the majority of samples. Results showed 14% loss of amplifiable mRNA per year, corresponding to a half-life of 4.6 years. The 4 most stable expressed genes were CALM2, RPL37A, ACTB, and RPLP0. Several of the other examined genes showed considerably instability over time (GAPDH, PSMC4, OAZ1, IPO8). In conclusion, we identified 4 genes robustly expressed over time and independent of neoplastic tissue content in the FFPE block. Other widely used reference genes were concluded to be less suited for retrospective analysis of FFPE breast samples. © 2013 by Lippincott Williams & Wilkins.