PMID- 28763741
OWN - NLM
STAT- MEDLINE
DCOM- 20171120
LR  - 20181202
IS  - 1879-1514 (Electronic)
IS  - 0166-445X (Linking)
VI  - 190
DP  - 2017 Sep
TI  - Expression stability and selection of optimal reference genes for gene expression 
      normalization in early life stage rainbow trout exposed to cadmium and copper.
PG  - 217-227
LID - S0166-445X(17)30202-3 [pii]
LID - 10.1016/j.aquatox.2017.07.009 [doi]
AB  - Gene expression analysis represents a powerful approach to characterize the 
      specific mechanisms by which contaminants interact with organisms. One of the key 
      considerations when conducting gene expression analyses using quantitative 
      real-time reverse transcription-polymerase chain reaction (qPCR) is the selection 
      of appropriate reference genes, which is often overlooked. Specifically, to reach 
      meaningful conclusions when using relative quantification approaches, expression 
      levels of reference genes must be highly stable and cannot vary as a function of 
      experimental conditions. However, to date, information on the stability of 
      commonly used reference genes across developmental stages, tissues and after 
      exposure to contaminants such as metals is lacking for many vertebrate species 
      including teleost fish. Therefore, in this study, we assessed the stability of 
      expression of 8 reference gene candidates in the gills and skin of three 
      different early life-stages of rainbow trout after acute exposure (24h) to two 
      metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes 
      were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), 
      elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase 
      (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine 
      phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal 
      RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative 
      DeltaCt method were employed to systematically evaluate the expression stability of 
      these candidate genes under control and exposed conditions as well as across 
      three different life-stages. Finally, stability of genes was ranked by taking 
      geometric means of the ranks established by the different methods. Stability of 
      reference genes was ranked in the following order (from lower to higher 
      stability): HPRT<GAPDH<EF1a<G6PD<RPL8<DRP2<b-actin in gills of fish exposed to 
      Cd; b-actin<GAPDH<G6PD<DRP2<RPL8<HPRT<EF1a in gills of fish exposed to Cu; 
      RPL8<HPRT<GAPDH<G6PD<EF1a<DRP2<b-actin in the skin of fish exposed to Cd; and 
      EF1a<GAPDH<RPL8<HPRT<G6PD<b-actin<DRP2 in the skin of fish exposed to Cu. The 
      results demonstrated that the stability of reference genes depended on the metal, 
      life-stage and/or organ in question. Thus, attention should be paid to these 
      factors before selection of reference gene for relative quantification of the 
      gene expressions.
CI  - Copyright (c) 2017 Elsevier B.V. All rights reserved.
FAU - Shekh, Kamran
AU  - Shekh K
AD  - Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; 
      Toxicology Graduate Program, University of Saskatchewan, Saskatoon, SK, S7N 5B3, 
      Canada. Electronic address: kamran.shekh@gmail.com.
FAU - Tang, Song
AU  - Tang S
AD  - National Institute of Environmental Health, Chinese Center for Disease Control 
      and Prevention, Beijing, 100021, China.
FAU - Niyogi, Som
AU  - Niyogi S
AD  - Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; 
      Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E2, 
      Canada.
FAU - Hecker, Markus
AU  - Hecker M
AD  - Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; 
      School of Environment and Sustainability, University of Saskatchewan, Saskatoon, 
      SK, S7N 5C8, Canada.
LA  - eng
PT  - Journal Article
DEP - 20170724
PL  - Netherlands
TA  - Aquat Toxicol
JT  - Aquatic toxicology (Amsterdam, Netherlands)
JID - 8500246
RN  - 0 (Water Pollutants, Chemical)
RN  - 00BH33GNGH (Cadmium)
RN  - 789U1901C5 (Copper)
SB  - IM
MH  - Animals
MH  - Cadmium/*toxicity
MH  - Copper/*toxicity
MH  - Gene Expression Profiling/methods
MH  - Gills/drug effects/enzymology
MH  - Life Cycle Stages/*drug effects/genetics
MH  - Oncorhynchus mykiss/*genetics/growth & development
MH  - Real-Time Polymerase Chain Reaction/methods
MH  - Skin/drug effects/enzymology
MH  - Transcriptome/*drug effects
MH  - Water Pollutants, Chemical/*toxicity
OTO - NOTNLM
OT  - Cadmium
OT  - Copper
OT  - Gene expression
OT  - Life-stages
OT  - Rainbow trout
OT  - Real-time PCR
OT  - Reference genes
EDAT- 2017/08/02 06:00
MHDA- 2017/11/29 06:00
CRDT- 2017/08/02 06:00
PHST- 2017/04/28 00:00 [received]
PHST- 2017/07/15 00:00 [revised]
PHST- 2017/07/17 00:00 [accepted]
PHST- 2017/08/02 06:00 [pubmed]
PHST- 2017/11/29 06:00 [medline]
PHST- 2017/08/02 06:00 [entrez]
AID - S0166-445X(17)30202-3 [pii]
AID - 10.1016/j.aquatox.2017.07.009 [doi]
PST - ppublish
SO  - Aquat Toxicol. 2017 Sep;190:217-227. doi: 10.1016/j.aquatox.2017.07.009. Epub 
      2017 Jul 24.