Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage.
Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30 min anaesthetised with isoeugenol, after 30 min without anaesthesia, after 120 min anaesthetised with isoeugenol, after 120 min with 15 min anaesthesia with metacaine or after 120 min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT-PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30 min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120 min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30 min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.
Trösse
Christiane
C
National Institute of Nutrition and Seafood Research (NIFES), P.O. Box 2029, 5817, Nordnes, Bergen, Norway. ctr@nifes.no
Waagbø
Rune
R
Breck
Olav
O
Olsvik
Pål A
PA
D000777:Anesthetics / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D002386:Cataract / Q000378:metabolism / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D007908:Lens, Crystalline / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020317:Salmo salar; D013048:Specimen Handling / Q000379:methods / Q000662:veterinary; D018709:Statistics, Nonparametric; D013315:Stress, Psychological / Q000378:metabolism*; D014021:Tissue Preservation / Q000379:methods / Q000662:veterinary
D000777:Anesthetics; D017931:DNA Primers; D012313:RNA
Integr Comp Biol. 2002 Jul;42(3):517-25
21708747
Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
12184808
Exp Eye Res. 2005 May;80(5):709-25
15862178
Nucleic Acids Res. 2005 Mar 30;33(6):e56
15800207
J Fish Dis. 2005 Jun;28(6):357-71
15960659
Mol Vis. 2009 Jul 09;15:1332-50
19597568
Comp Biochem Physiol A Mol Integr Physiol. 2008 Apr;149(4):396-404
18308603
Mol Aspects Med. 2006 Apr-Jun;27(2-3):126-39
16469371
Prog Retin Eye Res. 2003 Sep;22(5):657-82
12892645
Annu Rev Biochem. 1995;64:97-112
7574505
Physiol Rev. 1997 Jul;77(3):591-625
9234959
BMC Mol Biol. 2006 Jan 31;7:3
16448564
Biochem J. 2002 Aug 15;366(Pt 1):45-55
12020353
Vet Ophthalmol. 2006 Sep-Oct;9(5):292-8
16939456
BMC Mol Biol. 2005 Nov 17;6:21
16293192
BMC Mol Biol. 2007 Jun 08;8:48
17559653