title: Identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer.
abstract: OBJECTIVE
  Housekeeping genes as endogenous references are generally used for the relative quantification of target genes in gene profiling studies. To date that issue has not been sufficiently investigated in bladder cancer. From a panel of 9 potential candidates we selected the most stable housekeeping genes for gene normalization in bladder cancer tissue.
  
  
  METHODS
  Expression profiles of the 9 genes ACTB, ALAS1, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, SDHA and TBP were established in matched malignant and nonmalignant tissue specimens from 14 patients with bladder cancer. Quality assessment of isolated RNA was performed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California) and real-time reverse transcriptase-polymerase chain reaction was performed with LightCycler. The software geNorm and NormFinder (Aarhus University, Aarhus, Denmark) were used to identify the most suitable reference genes.
  
  
  RESULTS
  RNA was isolated with high purity and integrity. Candidate reference genes showed a broad range of between 20 and 34 polymerase chain reaction cycles. Expression did not depend on patient sex or tumor stage. GAPD, G6PD and HMBS showed significant differences in expression between malignant and nonmalignant pairs (at least p <0.04). Expression of the remaining genes did not differ between the matched pairs. SDHA and TBP were the most stably expressed genes, covering higher and lower expression levels.
  
  
  CONCLUSIONS
  For normalization purposes in gene profiling studies of bladder cancer the genes SDH and TBP are recommended as single reference genes depending on the expression level of the target gene or more favorably in combination.
authors.affiliation: Department of Urology and Clinic of Internal Medicine II (Oncology and Hematology), Charité-University Medicine Berlin, Berlin, Germany.
authors.forename: Falk
authors.initials: F
authors.lastname: Ohl
authors.forename: Monika
authors.initials: M
authors.lastname: Jung
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Radonić
authors.forename: Markus
authors.initials: M
authors.lastname: Sachs
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Loening
authors.forename: Klaus
authors.initials: K
authors.lastname: Jung
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020043:Genes, Essential / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000235:genetics*