3 Populations and Pathways
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3.1.9 RNA Studies: Q-PCR
Q-PCR stands for “Quantitative PCR”, also sometimes known as “Real-Time PCR”.
With this technique, the gene of interest is assayed in control and “treated” animals
using fluorescently labelled primers (often SYBR Green) and incorporation of these
primers is monitored during the PCR run by laser excitation. The level of fluorescence directly reflects the amount of PCR product generated. By comparing the
point in the PCR reaction where the amplification enters the log phase for both control and treated samples and determining the difference between the two, a measure
of the change in relative levels of transcript abundance caused by the treatment can
be made. There are a number of ways to control and validate this technique (see
Pfaffl 2001, Pfaffl et al. 2002, Radonic et al. 2004). This technique is often used
in tandem with microarray analyses, as the estimate of the change in expression
level produced via Q-PCR is more accurate than that produced via microarray analysis. However, designing and testing specific primers is a time-consuming process.
Microarrays are therefore best adapted for the initial, global overview of transcriptional activity, whereas Q-PCR is used subsequently to analyse the expression of
specific candidate genes identified by the microarray analysis.
In the previous sections, the main tools that are available for ecological genomics
studies have been briefly described. Note however, that this list of tools is continually expanding, fuelled by technological spin-offs from the Human Genome Project.
It is now important to outline some of their uses in the marine environment and how
they can contribute to our knowledge on population dynamics, biodiversity, and
environmental issues.
3.2 Population Genomics
Population genomics differs from population genetics in that it involves significantly increased coverage of the genome and the use of bioinformatics facilities to
deliver molecular tools as well as to analyze large datasets. Both neutral and selected
markers are targeted in population genomics in order to simultaneously analyze and
discriminate demographic (i.e. change in population size) and selective processes
(i.e. effects of environmental constraints).
It combines the methodology developments derived from genome analyses with
the conceptual framework of population genetics (for a review see Luikart et al.
2003). It can be broadly defined as the screening and analysis of a large number of DNA regions in order to address evolutionary or ecological issues (Black
et al. 2001, Luikart et al. 2003, Schlotterer 2003, Feder and Mitchells-Olds 2003).
Methodologies and approaches are highly interdisciplinary. For example, it is obvious that any relationship between a phenotype and an environment can be tracked
to both the level of the population or of the individual in order to define the
genetic basis of this phenotype. However, this relationship can also be analysed
from a more functional perspective by examining a set of co-expressed genes
83
3.1.9 RNA Studies: Q-PCR
Q-PCR stands for “Quantitative PCR”, also sometimes known as “Real-Time PCR”.
With this technique, the gene of interest is assayed in control and “treated” animals
using fluorescently labelled primers (often SYBR Green) and incorporation of these
primers is monitored during the PCR run by laser excitation. The level of fluorescence directly reflects the amount of PCR product generated. By comparing the
point in the PCR reaction where the amplification enters the log phase for both control and treated samples and determining the difference between the two, a measure
of the change in relative levels of transcript abundance caused by the treatment can
be made. There are a number of ways to control and validate this technique (see
Pfaffl 2001, Pfaffl et al. 2002, Radonic et al. 2004). This technique is often used
in tandem with microarray analyses, as the estimate of the change in expression
level produced via Q-PCR is more accurate than that produced via microarray analysis. However, designing and testing specific primers is a time-consuming process.
Microarrays are therefore best adapted for the initial, global overview of transcriptional activity, whereas Q-PCR is used subsequently to analyse the expression of
specific candidate genes identified by the microarray analysis.
In the previous sections, the main tools that are available for ecological genomics
studies have been briefly described. Note however, that this list of tools is continually expanding, fuelled by technological spin-offs from the Human Genome Project.
It is now important to outline some of their uses in the marine environment and how
they can contribute to our knowledge on population dynamics, biodiversity, and
environmental issues.
3.2 Population Genomics
Population genomics differs from population genetics in that it involves significantly increased coverage of the genome and the use of bioinformatics facilities to
deliver molecular tools as well as to analyze large datasets. Both neutral and selected
markers are targeted in population genomics in order to simultaneously analyze and
discriminate demographic (i.e. change in population size) and selective processes
(i.e. effects of environmental constraints).
It combines the methodology developments derived from genome analyses with
the conceptual framework of population genetics (for a review see Luikart et al.
2003). It can be broadly defined as the screening and analysis of a large number of DNA regions in order to address evolutionary or ecological issues (Black
et al. 2001, Luikart et al. 2003, Schlotterer 2003, Feder and Mitchells-Olds 2003).
Methodologies and approaches are highly interdisciplinary. For example, it is obvious that any relationship between a phenotype and an environment can be tracked
to both the level of the population or of the individual in order to define the
genetic basis of this phenotype. However, this relationship can also be analysed
from a more functional perspective by examining a set of co-expressed genes