Analysis of transcriptional responses of normalizing genes on Crassostrea brasiliana under different experimental conditions.
Bivalves show remarkable plasticity to environmental changes and have been proposed as sentinel organisms in biomonitoring. Studies related to transcriptional analysis using quantitative real-time polymerase chain reaction (qRT-PCR) in these organisms have notably increased, imposing a need to identify and validate adequate reference genes for an accurate and reliable analysis. In the present study, 9 reference genes were selected from transcriptome data of Crassostrea brasiliana to identify their suitability as qRT-PCR normalizer genes. The transcriptional patterns were analyzed in gills of oysters under 3 different conditions: different temperatures (18, 24, or 32 °C) and phenanthrene (100 µg L-1) combined exposure; different salinities (10, 25, or 35‰) and phenanthrene combined exposure; and 10% of diesel fuel water-accommodated fraction (diesel-WAF) exposure. Reference gene stability was calculated using 5 algorithms (geNorm, NormFinder, BestKeeper, ΔCt, RefFinder). Transcripts of ankyrin-like (ANK), glyceraldehyde 3-phosphate dehydrogenase-like (GAPDH), and α-tubulin-like (TUBA) genes showed minor changes in different temperature/phenanthrene treatment. Transcripts of ANK, β-actin-like, and β-tubulin-like genes showed better stability at salinity/phenanthrene treatment, and ANK, TUBA, and 28S ribosomal protein-like genes showed the most stable transcription pattern in oysters exposed to diesel-WAF exposure. The present study constitutes the first systematic analysis of reference gene selection for qRT-PCR normalization in C. brasiliana. These genes could be employed in studies using qRT-PCR analysis under similar experimental conditions. Environ Toxicol Chem 2017;36:2190-2198. © 2017 SETAC.
Biochemistry Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Gabrielle do Amaral E Silva
GDAES
Müller
Biochemistry Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Daína
D
de Lima
Aquaculture Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Flávia Lucena
FL
Zacchi
Biochemistry Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Rômi Sharon
RS
Piazza
Fishery Engineering Department, Santa Catarina State University, Laguna, Santa Catarina, Brazil.
Karim Hahn
KH
Lüchmann
Biochemistry Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Jacó Joaquim
JJ
Mattos
Department of Environmental Sciences, University of California, Riverside California, United States.
Daniel
D
Schlenk
Biochemistry Department, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Afonso Celso Dias
ACD
Bainy
D000818:Animals; D017487:Ankyrins / Q000235:genetics; D049896:Crassostrea / Q000187:drug effects / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods*; D005742:Gasoline / Q000633:toxicity; D020869:Gene Expression Profiling; D005880:Gills / Q000187:drug effects / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D010616:Phenanthrenes / Q000633:toxicity; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D054712:Salinity; D013696:Temperature; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D014404:Tubulin / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D017487:Ankyrins; D005742:Gasoline; D010616:Phenanthrenes; D014404:Tubulin; D014874:Water Pollutants, Chemical; C031181:phenanthrene; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Aquatic invertebrates; Biomarkers; Environmental toxicology; Mollusc toxicology; Polycyclic Aromatic hydrocarbons