242
M.L. Cancela et al.
β-actin, a supposed housekeeping gene and as such frequently used as a reference
gene in qPCR assays, was also reported to be amongst the differentially expressed
transcripts suggesting that its use under these circumstances may not be adequate.
In this study, the authors also reported the stimulation of several signalling pathways upon infection, as well as the existence of significant differences between two
closely related species, the blue and the channel catfish. Most of the transcripts differentially expressed between those two species belong to the MHC class I pathway.
More recently, microarray profiling was used to characterize the immune response
of the yellow tail Seriola quinquerdiata upon exposure to immunostimulants such
as ConA and lipopolysaccharide (LPS; Darawiroj et al. 2008). Similar studies have
been performed on the Atlantic salmon (Ewart et al. 2005, Martin et al. 2006) and
the rainbow trout (Tilton et al. 2005, Gerwick et al. 2007). Finally, MacKenzie et al.
(2008) have shown in the rainbow trout that it is possible to distinguish the immune
response against two different immune agents, i.e. a virus (IHNV) and a bacterial
cell wall component, using microarray gene expression profiling.
7.6.2.3 Real-Time PCR to Identify Candidate Markers for Disease Detection
The polymerase chain reaction (PCR), first introduced by Kleppe et al. (1971) and
further developed by Mullis and Faloona (1987) allows detection of a given transcript following amplification of a specific part of the genome. The first application
using PCR for quantifications was introduced by Higuchi et al. (1993) 10 years
later. Today quantitative real-time PCR (qPCR) is commonly used in human diagnostics and in expression studies of various biological systems (Bustin et al. 2005).
Accuracy of qPCR depends on factors such as RNA template quality, type of polymerase, primers, reference genes used and data analysis. qPCR has been used in
several studies to reveal the role of immune-related genes in the immune response
of economically important fish species. For example, qPCR analysis revealed the
role of hepcidin, an antimicrobial peptide, in the innate immunity of the gilthead
seabream following bacterial infection (Cuesta et al. 2008). Raida and Buchmann
(2007) showed by qPCR that expression of both innate and adaptive immune
response genes is temperature-dependent in the trout. qPCR has also been used to
characterize virulence mechanisms (Sepulcre et al. 2007), prophylaxis and treatment
of viral and bacterial-related diseases (Samuelsen et al. 2006).
7.6.3 How Can Genetic Linkage, RH and Physical Maps
Contribute to Shedding Light on Fish–Pathogen
Interactions?
Besides growth and stress responses, economically important traits for farming of
most fish also include immune response and host–pathogen interactions. Regions
of the genome corresponding to QTL have been identified and, in some cases,
the molecular polymorphism underlying the QTL has been identified in terrestrial
M.L. Cancela et al.
β-actin, a supposed housekeeping gene and as such frequently used as a reference
gene in qPCR assays, was also reported to be amongst the differentially expressed
transcripts suggesting that its use under these circumstances may not be adequate.
In this study, the authors also reported the stimulation of several signalling pathways upon infection, as well as the existence of significant differences between two
closely related species, the blue and the channel catfish. Most of the transcripts differentially expressed between those two species belong to the MHC class I pathway.
More recently, microarray profiling was used to characterize the immune response
of the yellow tail Seriola quinquerdiata upon exposure to immunostimulants such
as ConA and lipopolysaccharide (LPS; Darawiroj et al. 2008). Similar studies have
been performed on the Atlantic salmon (Ewart et al. 2005, Martin et al. 2006) and
the rainbow trout (Tilton et al. 2005, Gerwick et al. 2007). Finally, MacKenzie et al.
(2008) have shown in the rainbow trout that it is possible to distinguish the immune
response against two different immune agents, i.e. a virus (IHNV) and a bacterial
cell wall component, using microarray gene expression profiling.
7.6.2.3 Real-Time PCR to Identify Candidate Markers for Disease Detection
The polymerase chain reaction (PCR), first introduced by Kleppe et al. (1971) and
further developed by Mullis and Faloona (1987) allows detection of a given transcript following amplification of a specific part of the genome. The first application
using PCR for quantifications was introduced by Higuchi et al. (1993) 10 years
later. Today quantitative real-time PCR (qPCR) is commonly used in human diagnostics and in expression studies of various biological systems (Bustin et al. 2005).
Accuracy of qPCR depends on factors such as RNA template quality, type of polymerase, primers, reference genes used and data analysis. qPCR has been used in
several studies to reveal the role of immune-related genes in the immune response
of economically important fish species. For example, qPCR analysis revealed the
role of hepcidin, an antimicrobial peptide, in the innate immunity of the gilthead
seabream following bacterial infection (Cuesta et al. 2008). Raida and Buchmann
(2007) showed by qPCR that expression of both innate and adaptive immune
response genes is temperature-dependent in the trout. qPCR has also been used to
characterize virulence mechanisms (Sepulcre et al. 2007), prophylaxis and treatment
of viral and bacterial-related diseases (Samuelsen et al. 2006).
7.6.3 How Can Genetic Linkage, RH and Physical Maps
Contribute to Shedding Light on Fish–Pathogen
Interactions?
Besides growth and stress responses, economically important traits for farming of
most fish also include immune response and host–pathogen interactions. Regions
of the genome corresponding to QTL have been identified and, in some cases,
the molecular polymorphism underlying the QTL has been identified in terrestrial