title: Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is β-actin a reliable normalizer for photoaging studies?
abstract: Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm(2) UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.
authors.affiliation: Dipartimento Scienze Cliniche Specialistiche ed Odotontostomatologiche, Università Politecnica delle Marche, I-60131 Ancona, Italy. f.bruge@libero.it
authors.forename: F
authors.initials: F
authors.lastname: Brugè
authors.forename: E
authors.initials: E
authors.lastname: Venditti
authors.forename: L
authors.initials: L
authors.lastname: Tiano
authors.forename: G P
authors.initials: GP
authors.lastname: Littarru
authors.forename: E
authors.initials: E
authors.lastname: Damiani
mesh_terms: D000199:Actins / Q000235:genetics*; D015687:Cell Hypoxia / Q000235:genetics / Q000528:radiation effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology; D014466:Ultraviolet Rays
chemical_list: D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D020781:Matrix Metalloproteinase 1