360
V. Mittard-Runte et al.
nucleotides are used to synthesise longer oligonucleotides directly on the substrate.
One of the most important parameters is the length of the probe sequences that
can be deposited or synthesized. Sequence lengths can vary from a few hundred
base-pairs, when cDNA is spotted, to short oligonucleotides. Commercial arrays
are normally produced using short oligonucleotides. Affymetrix
R
arrays, for example have multiple different probe sequences per gene of 25 bp in length. Combining
measurements made with several different probes is necessary to compensate for
non specific binding of target DNA. Other oligonucleotide arrays contain oligos of
between 50 and 80 bp, which tend to be more specific. Another important difference
between microarray platforms is the number of channels they support. Two-colour
microarrays support direct comparison of two experimental conditions per array.
Single channel arrays can be used for single conditions or for a comparative
study using multiple arrays. Furthermore, commercial array providers have different
set-up costs and times for individual array designs and the number of features they
support.
9.4.1.1 Variation and Replication
As with any other biological experiment, microarray experiments will show a certain
amount of measurement variability and measurement error. Sources of variation in
a microarray experiment can be divided into technical and biological variation.
Deviations in the technical process can have a large influence on the results of
microarray experiments. The extent of these influences depends on the technology
and microarray platform used and can be observed in replicated experiments in the
same laboratory and also between laboratories. Primary causes are variations in the
application of protocols. Further technical variation stems from the microarray production process such as variations in feature sizes and concentrations (Bammler
et al. 2005). Some studies have also shown the large impact of differential probe
sequences as a source of cross-platform variation. Other technical problems include
scanner settings, as well as image segmentation and quantification (Yauk et al. 2004,
Repsilber and Ziegler 2005, Yauk et al. 2005).
Even assuming perfect measurement technology, there would still be biological
variation within the organism, either between different cells or between individuals.
This variation is due to the individual genetic characteristics of the organisms and
also due to variations in environmental conditions. The level of biological variation
is assessed by performing the experiment multiple times under the same conditions
and by harvesting several samples. This is termed replication. In general, biological variation seems to be a more important factor than technical variation for the
assessment of the significance of the results obtained. Biological replications are
preferable to technical replications (Allison et al. 2006).
Biological replicates will be influenced by both technical and biological variation, thereby serving to assess the overall variability of the experiment. If the number
of replications exceeds the number of available microarrays, a pooling procedure is
often used to generate a mixture of samples.
V. Mittard-Runte et al.
nucleotides are used to synthesise longer oligonucleotides directly on the substrate.
One of the most important parameters is the length of the probe sequences that
can be deposited or synthesized. Sequence lengths can vary from a few hundred
base-pairs, when cDNA is spotted, to short oligonucleotides. Commercial arrays
are normally produced using short oligonucleotides. Affymetrix
R
arrays, for example have multiple different probe sequences per gene of 25 bp in length. Combining
measurements made with several different probes is necessary to compensate for
non specific binding of target DNA. Other oligonucleotide arrays contain oligos of
between 50 and 80 bp, which tend to be more specific. Another important difference
between microarray platforms is the number of channels they support. Two-colour
microarrays support direct comparison of two experimental conditions per array.
Single channel arrays can be used for single conditions or for a comparative
study using multiple arrays. Furthermore, commercial array providers have different
set-up costs and times for individual array designs and the number of features they
support.
9.4.1.1 Variation and Replication
As with any other biological experiment, microarray experiments will show a certain
amount of measurement variability and measurement error. Sources of variation in
a microarray experiment can be divided into technical and biological variation.
Deviations in the technical process can have a large influence on the results of
microarray experiments. The extent of these influences depends on the technology
and microarray platform used and can be observed in replicated experiments in the
same laboratory and also between laboratories. Primary causes are variations in the
application of protocols. Further technical variation stems from the microarray production process such as variations in feature sizes and concentrations (Bammler
et al. 2005). Some studies have also shown the large impact of differential probe
sequences as a source of cross-platform variation. Other technical problems include
scanner settings, as well as image segmentation and quantification (Yauk et al. 2004,
Repsilber and Ziegler 2005, Yauk et al. 2005).
Even assuming perfect measurement technology, there would still be biological
variation within the organism, either between different cells or between individuals.
This variation is due to the individual genetic characteristics of the organisms and
also due to variations in environmental conditions. The level of biological variation
is assessed by performing the experiment multiple times under the same conditions
and by harvesting several samples. This is termed replication. In general, biological variation seems to be a more important factor than technical variation for the
assessment of the significance of the results obtained. Biological replications are
preferable to technical replications (Allison et al. 2006).
Biological replicates will be influenced by both technical and biological variation, thereby serving to assess the overall variability of the experiment. If the number
of replications exceeds the number of available microarrays, a pooling procedure is
often used to generate a mixture of samples.