title: Validating reference genes within a mouse model system of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity.
abstract: BACKGROUND
  Quantitative real-time PCR (qPCR) is the "gold-standard" technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes.
  
  
  RESULTS
  We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized.
  
  
  CONCLUSIONS
  We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets.
authors.affiliation: Informatics and Bio-Computing Platform, Ontario Institute for Cancer Research, Toronto, Canada.
authors.forename: Stephenie D
authors.initials: SD
authors.lastname: Prokopec
authors.forename: Nicholas B
authors.initials: NB
authors.lastname: Buchner
authors.forename: Natalie S
authors.initials: NS
authors.lastname: Fox
authors.forename: Lauren C
authors.initials: LC
authors.lastname: Chong
authors.forename: Denise Y F
authors.initials: DY
authors.lastname: Mak
authors.forename: John D
authors.initials: JD
authors.lastname: Watson
authors.forename: Arturas
authors.initials: A
authors.lastname: Petronis
authors.forename: Raimo
authors.initials: R
authors.lastname: Pohjanvirta
authors.forename: Paul C
authors.initials: PC
authors.lastname: Boutros
mesh_terms: D000465:Algorithms; D000818:Animals; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D020043:Genes, Essential / Q000187:drug effects*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D015999:Multivariate Analysis; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D020648:Peptide Elongation Factor 1; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase
keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin; AHR; ARNT; C(q); Mouse model; NEL; Normalization; Quantitative real-time PCR; Reference genes; TCDD; aryl hydrocarbon receptor; aryl hydrocarbon receptor nuclear translocator; mRNA abundance; normalized expression level; qPCR; quantification cycle; quantitative real-time PCR