Journal
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY
Volume 153, Issue 3, Pages 259-268Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cbpc.2010.11.009
Keywords
Reference genes; Internal control; Real-time PCR; EDCs; Biomarker
Funding
- Ministry of Land, Transportation and Maritime Affairs, Republic of Korea
- MKE (The Ministry of Knowledge Economy), Korea, under the ITRC (Information Technology Research Center) [NIPA-2009-C1090-0903-0007]
- Korea Institute of Marine Science & Technology Promotion (KIMST) [20046001] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
- Ministry of Public Safety & Security (MPSS), Republic of Korea [C1090-1121-0015] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
- National Research Foundation of Korea [핵09A2518] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 17 beta-estradiol (E2). The expression levels of several conventional HKGs, such as 18s rRNA, glyceraldehyde-3-phosphate dehydrogenase and beta-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. (C) 2010 Elsevier Inc. All rights reserved.
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