title: Reference gene validation for RT-qPCR, a note on different available software packages.
abstract: BACKGROUND
  An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms.
  
  
  RESULTS
  14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account.
  
  
  CONCLUSIONS
  This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.
authors.affiliation: Ghent University, Department of Internal Medicine, Ghent, Belgium.
authors.forename: Ward
authors.initials: W
authors.lastname: De Spiegelaere
authors.affiliation: Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Giessen, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Dern-Wieloch
authors.affiliation: Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Giessen, Germany.
authors.forename: Roswitha
authors.initials: R
authors.lastname: Weigel
authors.affiliation: Harvard Medical School, Department of Pediatrics, Boston, Massachusetts, United States of America.
authors.forename: Valérie
authors.initials: V
authors.lastname: Schumacher
authors.affiliation: University of Bonn Medical School, Department of Developmental Pathology, Bonn, Germany.
authors.forename: Hubert
authors.initials: H
authors.lastname: Schorle
authors.affiliation: University of Bonn Medical School, Department of Developmental Pathology, Bonn, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettersheim
authors.affiliation: Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Giessen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Bergmann
authors.affiliation: University of Veterinary Medicine Hannover, Institute of Anatomy, Hannover, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Brehm
authors.affiliation: University of Münster, Center of Andrology and Reproductive Medicine, Münster, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kliesch
authors.affiliation: Ghent University, Department of Internal Medicine, Ghent, Belgium.
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
authors.affiliation: Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Giessen, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Fink
mesh_terms: D000465:Algorithms; D002460:Cell Line; D018920:Coculture Techniques; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012984:Software
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