title: Validation of housekeeping gene and impact on normalized gene expression in clear cell renal cell carcinoma: critical reassessment of YBX3/ZONAB/CSDA expression.
abstract: BACKGROUND
  YBX3/ZONAB/CSDA is an epithelial-specific transcription factor acting in the density-based switch between proliferation and differentiation. Our laboratory reported overexpression of YBX3 in clear cell renal cell arcinoma (ccRCC), as part of a wide study of YBX3 regulation in vitro and in vivo. The preliminary data was limited to 5 cases, of which only 3 could be compared to paired normal tissue, and beta-Actin was used as sole reference to normalize gene expression. We thus decided to re-evaluate YBX3 expression by real-time-PCR in a larger panel of ccRCC samples, and their paired healthy tissue, with special attention on experimental biases such as inter-individual variations, primer specificity, and reference gene for normalization.
  
  
  RESULTS
  Gene expression was measured by RT-qPCR in 16 ccRCC samples, each compared to corresponding healthy tissue to minimize inter-individual variations. Eight potential housekeeping genes were evaluated for expression level and stability among the 16-paired samples. Among tested housekeeping genes, PPIA and RPS13, especially in combination, proved best suitable to normalize gene expression in ccRCC tissues as compared to classical reference genes such as beta-Actin, GAPDH, 18S or B2M. Using this pair as reference, YBX3 expression level among a collection of 16 ccRCC tumors was not significantly increased as compared to normal adjacent tissues. However, stratification according to Fuhrman grade disclosed higher YBX3 expression levels in low-grade tumors and lower in high-grade tumors. Immunoperoxidase confirmed homogeneous nuclear staining for YBX3 in low-grade but revealed nuclear heterogeneity in high-grade tumors.
  
  
  CONCLUSIONS
  This paper underlines that special attention to reference gene products in the design of real-time PCR analysis of tumoral tissue is crucial to avoid misleading conclusions. Furthermore, we found that global YBX3/ZONAB/CSDA mRNA expression level may be considered within a "signature" of RCC grading.
authors.affiliation: CELL Unit, de Duve Institute and Université catholique de Louvain UCL-ICP, Avenue Hippocrate 75, 1200 Brussels, Belgium. sebastien.dupasquier@uclouvain.be.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dupasquier
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Delmarcelle
authors.forename: Etienne
authors.initials: E
authors.lastname: Marbaix
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cosyns
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Courtoy
authors.forename: Christophe E
authors.initials: CE
authors.lastname: Pierreux
mesh_terms: D000199:Actins / Q000235:genetics; D000328:Adult; D000368:Aged; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D002292:Carcinoma, Renal Cell / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D012269:Ribosomal Proteins / Q000235:genetics
chemical_list: D000199:Actins; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; C047013:RPS13 protein, human; D012269:Ribosomal Proteins; C094867:YBX3 protein, human