title: Validation of housekeeping genes as internal controls for studying biomarkers of endocrine-disrupting chemicals in disk abalone by real-time PCR.
abstract: Our experiments were designed to identify suitable housekeeping genes (HKGs) in disk abalone as internal controls to quantify biomarker expression following endocrine disrupting chemicals (EDCs). Relative expression levels of twelve candidate HKGs were examined by real-time reverse transcription PCR (qRT-PCR) in gill and hepatopancreas of abalone following a 7-day challenge with either tributyltin chloride (TBT) or 17β-estradiol (E2). The expression levels of several conventional HKGs, such as 18s rRNA, glyceraldehyde-3-phosphate dehydrogenase and β-actin, were significantly altered by the challenges, indicating that they might not be suitable internal controls. Instead, the geNorm analysis pinpointed ribosomal protein L-5/ elongation factor 1 and ribosomal protein L-5/ succinate dehydrogenase as the most stable HKGs under TBT and E2 challenges, respectively. Moreover, these three HKGs also showed the highest stabilities overall amongst different tissues, genders and EDC challenges. The expression of a biomarker gene, cytochrome P450 4B (CYP4), was also investigated and exhibited a significant increase after the challenges. Importantly, when unsuitable HKGs were used for normalization, the influence of two EDCs on CYP4 expression was imprecisely overestimated or underestimated, which strongly emphasized the importance of selecting appropriately validated HKGs as internal controls in biomarker studies.
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju, 690-756, Republic of Korea.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wan
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Cheol Young
authors.initials: CY
authors.lastname: Choi
authors.forename: Jae-Seong
authors.initials: JS
authors.lastname: Lee
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D042926:Cytochrome P-450 CYP4A / Q000378:metabolism; D052244:Endocrine Disruptors / Q000633:toxicity*; D004958:Estradiol / Q000633:toxicity*; D004967:Estrogens / Q000633:toxicity*; D005260:Female; D049851:Gastropoda / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D020043:Genes, Essential; D005880:Gills / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D043143:Hepatopancreas / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008297:Male; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014220:Trialkyltin Compounds / Q000633:toxicity*; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D015415:Biomarkers; D052244:Endocrine Disruptors; D004967:Estrogens; D014220:Trialkyltin Compounds; D014874:Water Pollutants, Chemical; D004958:Estradiol; C011559:tributyltin; D042926:Cytochrome P-450 CYP4A