title: Identification of reference proteins for Western blot analyses in mouse model systems of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity.
abstract: Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1) TCDD-induced fold-difference in protein content from basal levels, 2) inter- and intra- animal stability, and 3) the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2-3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed.
authors.affiliation: Informatics and Bio-computing Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada.
authors.forename: Stephenie D
authors.initials: SD
authors.lastname: Prokopec
authors.affiliation: Informatics and Bio-computing Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada.
authors.forename: John D
authors.initials: JD
authors.lastname: Watson
authors.affiliation: Laboratory of Toxicology, National Institute for Health and Welfare, Kuopio, Finland; Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland.
authors.forename: Raimo
authors.initials: R
authors.lastname: Pohjanvirta
authors.affiliation: Informatics and Bio-computing Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacology & Toxicology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Boutros
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D020543:Proteome / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards
chemical_list: D000072317:Polychlorinated Dibenzodioxins; D020543:Proteome; D012333:RNA, Messenger