Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).
Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.
Department of Veterinary Medicine, National Chiayi University, Chiayi, 600, Taiwan (R.O.C.).
I-Hua
IH
Chen
Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, 106, Taiwan (R.O.C.).
Lien-Siang
LS
Chou
Department of Veterinary Medicine, National Chiayi University, Chiayi, 600, Taiwan (R.O.C.).
Shih-Jen
SJ
Chou
Department of Veterinary Medicine, National Chiayi University, Chiayi, 600, Taiwan (R.O.C.).
Jiann-Hsiung
JH
Wang
School of Veterinary Medicine, University of California, Davis, California, 95616, United States of America.
Jeffrey
J
Stott
School of Veterinary Medicine, University of California, Davis, California, 95616, United States of America.
Myra
M
Blanchard
Farglory Ocean Park, Hualien, 604, Taiwan (R.O.C.).
I-Fan
IF
Jen
Department of Veterinary Medicine, National Chiayi University, Chiayi, 600, Taiwan (R.O.C.).
Wei-Cheng
WC
Yang
D000818:Animals; D050681:Bottle-Nosed Dolphin / Q000097:blood*; D020869:Gene Expression Profiling / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards
D012333:RNA, Messenger