We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, GAPDH and 18S rRNA, for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. While no overall trends were observed in the expression of the 18S rRNA, GAPDH was upregulated in melanoma tissue and cultured cells compared to the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing mRNA expression via GAPDH or 18S rRNA housekeeping genes.The number of skin cancer cases has been steadily increasing with the risk of developing malignant cutaneous melanoma doubling every 10-15 years [1]. However, in 2007 the new cases of melanoma was estimated at 59,940, nearly double that of previous estimates [2]. If melanoma is detected early (localized), the five year survival rate is ∼99% [3]. However, distant stage (metastatic) melanoma has a very poor prognosis (∼15% five year survival) [3]. Thus, there is a critical requirement for accurate assessment of melanoma initiation and progression, as well as the characterization of the more malignant later stages.The most reliable prognostic factor of melanoma is still the tumor thickness [4], thus underscoring the importance of early detection and surgery. Better knowledge of the underlying molecular mechanisms of transformation and melanoma progression would possibly aid in the discovery of novel biomarkers, potential therapeutic targets, or biochemical pathways. Previous studies have shown that gene expression profiling of melanoma mRNA levels may lead to novel molecular classification methods by identifying the subtypes of disease and possibly predicting phenotypic characteristics ([5] and references cited therein). Microarray analysis can be used for the high-throughput analysis of global gene expression levels, studying hundreds or thousands of targets at once. Quantitative real-time PCR (qRT-PCR or qPCR) allows for the sensitivity to analyze a narrow range of targets or to confirm previously obtained microarray data.Currently there are two quantification methods available for qPCR, absolute and relative. In the case of absolute quantification, genomic DNA or plasmids containing cloned qPCR target *Corresponding author. Department of Chemistry & Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991, USA. Tel: 1-561-297-2093. Fax: 1-561-297-2759. E-mail address: fieldsg@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal di...