An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV.
BACKGROUND
Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.


RESULTS
The expression stability of five commonly used housekeeping genes [beta-actin (ACTB), elongation factor 1-alpha (EF1A), ubiquitin (UBQ), glyceraldehyd-3-phosphate dehydrogenase (GAPDH) and tubulin alpha (TUBA)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium Piscirickettsia salmonis and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, UBQ and EF1A appeared as the most stable, although EF1A was slightly upregulated at late stages of P. salmonis infection in RTS11. ACTB instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of GAPDH and TUBA was also demonstrated.


CONCLUSIONS
Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of UBQ, ACTB and EF1A as reference genes in qRT-PCR assays for studying the effect of P. salmonis and IPNV on the host immune response.
Peña
Andrea A
AA
Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Av, Brasil 2950, Valparaíso, Chile. andrea.pena@ucv.cl.
Bols
Niels C
NC
Marshall
Sergio H
SH
Mar Biotechnol (NY). 2006 Jul-Aug;8(4):398-408
16676145
Virol J. 2005 Feb 10;2:7
15705200
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W43-6
17452344
Mol Immunol. 2008 Mar;45(5):1439-48
17928055
Genome Biol. 2007;8(2):R19
17291332
BMC Mol Biol. 2005 Nov 17;6:21
16293192
Comp Biochem Physiol B Biochem Mol Biol. 2008 May;150(1):23-32
18302990
J Cell Biochem. 2009 Oct 15;108(3):631-7
19681041
BMC Mol Biol. 2008 Jan 29;9:18
18230138
Antiviral Res. 1996 Mar;29(2-3):309-12
8739609
Appl Environ Microbiol. 1998 Aug;64(8):3066-9
9687475
J Mol Endocrinol. 2002 Aug;29(1):23-39
12200227
Vet Immunol Immunopathol. 2008 Apr 15;122(3-4):312-7
18272235
Cancer Res. 2004 Aug 1;64(15):5245-50
15289330
Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
12184808
Virus Res. 2007 Jun;126(1-2):159-71
17391795
Anal Biochem. 2005 Sep 1;344(1):141-3
16054107
Mol Immunol. 2006 Mar;43(8):1194-201
16139890
BMC Mol Biol. 2008 Nov 12;9:102
19014500
Fish Shellfish Immunol. 2005 Apr;18(4):279-95
15561559
Genes Immun. 2005 Jun;6(4):279-84
15815687
Biotechnol Lett. 2004 Mar;26(6):509-15
15127793
J Biochem Biophys Methods. 2000 Nov 20;46(1-2):69-81
11086195
Virol J. 2007 Dec 03;4:130
18053162
Biochim Biophys Acta. 1999 Jul 13;1432(2):159-84
10407139