9 Genomic Techniques and How to Apply Them to Marine Questions
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be applied. The resulting sequence reads can be mapped onto the genome and be
analysed quantitatively. This method also applies to prokaryotes, and allows for
a high coverage of complete transcripts. Despite its high potential, it has currently
only been used for a limited number of studies and clear guidelines for the statistical
analysis of shotgun-transcriptomics data still need to be developed.
9.4.1 Fundamentals of Microarray Technology
Microarrays allow the parallel measurement of the abundance of mRNA corresponding to thousands of genes (Lipshutz et al. 1995, Schena et al. 1995).
In consequence, microarrays are considered to be a high-throughput technique
(Lipshutz et al. 1999, Miron and Nadon 2006, Küster et al. 2007). Microarray
technology evolved rapidly during the late 1990s and this technology has marked
a turning point in functional genomics due to their wide range of applications and
relative cost-efficiency. A multitude of diverse array technologies, protocols for their
application, and statistical methods for data evaluation have been developed.
Despite the many technological differences at the detailed level, the common
principle of all microarray platforms is rather straightforward. DNA molecules with
a defined nucleotide sequences are attached to the surface of a solid support, usually coated glass. Molecules of the same type share a small region on the surface.
These regions are called features or spots and are arranged in a grid pattern. Current
technology enables a density of more than 10,000 features per cm 2 . To measure messenger RNA, it has to be extracted and converted into cDNA by reverse transcription
using reverse transcriptase. The cDNA is labelled with a fluorescent marker allowing quantification of the number of DNA copies. Some protocols also allow the use
of mRNA directly, without reverse transcription. The solution of labelled molecules
(also called targets) is then brought into contact with the surface of the microarray. In a process of parallel hybridization, the labelled single-stranded RNA or
cDNA molecules hybridize with their single-stranded counterparts, representing the
nucleotide sequence of the complementary strand on the surface of the microarray.
The approximate number of target molecules bound to a given feature is measured
by a detection device. Often a laser scanner producing visible images is used. The
laser scanners excite fluorescent dyes with laser radiation of a defined bandwidth
and this results in the emission of light of a defined emission bandwidth. The resulting images are processed and transformed into intensity measurements by image
analysis software.
Many companies commercially produce microarrays. The technologies vary in
many details, for example the array substrate, and in the process of generating
the probe sequences. Spotted microarrays are produced by a deposition of small
amounts of DNA solution on the substrate by robotic spotters. Other companies (for example Agilent) use ink-jet technology to deposit DNA. Affymetrix
R
and NimbleGen arrays are produced using a photolithographic process in which
359
be applied. The resulting sequence reads can be mapped onto the genome and be
analysed quantitatively. This method also applies to prokaryotes, and allows for
a high coverage of complete transcripts. Despite its high potential, it has currently
only been used for a limited number of studies and clear guidelines for the statistical
analysis of shotgun-transcriptomics data still need to be developed.
9.4.1 Fundamentals of Microarray Technology
Microarrays allow the parallel measurement of the abundance of mRNA corresponding to thousands of genes (Lipshutz et al. 1995, Schena et al. 1995).
In consequence, microarrays are considered to be a high-throughput technique
(Lipshutz et al. 1999, Miron and Nadon 2006, Küster et al. 2007). Microarray
technology evolved rapidly during the late 1990s and this technology has marked
a turning point in functional genomics due to their wide range of applications and
relative cost-efficiency. A multitude of diverse array technologies, protocols for their
application, and statistical methods for data evaluation have been developed.
Despite the many technological differences at the detailed level, the common
principle of all microarray platforms is rather straightforward. DNA molecules with
a defined nucleotide sequences are attached to the surface of a solid support, usually coated glass. Molecules of the same type share a small region on the surface.
These regions are called features or spots and are arranged in a grid pattern. Current
technology enables a density of more than 10,000 features per cm 2 . To measure messenger RNA, it has to be extracted and converted into cDNA by reverse transcription
using reverse transcriptase. The cDNA is labelled with a fluorescent marker allowing quantification of the number of DNA copies. Some protocols also allow the use
of mRNA directly, without reverse transcription. The solution of labelled molecules
(also called targets) is then brought into contact with the surface of the microarray. In a process of parallel hybridization, the labelled single-stranded RNA or
cDNA molecules hybridize with their single-stranded counterparts, representing the
nucleotide sequence of the complementary strand on the surface of the microarray.
The approximate number of target molecules bound to a given feature is measured
by a detection device. Often a laser scanner producing visible images is used. The
laser scanners excite fluorescent dyes with laser radiation of a defined bandwidth
and this results in the emission of light of a defined emission bandwidth. The resulting images are processed and transformed into intensity measurements by image
analysis software.
Many companies commercially produce microarrays. The technologies vary in
many details, for example the array substrate, and in the process of generating
the probe sequences. Spotted microarrays are produced by a deposition of small
amounts of DNA solution on the substrate by robotic spotters. Other companies (for example Agilent) use ink-jet technology to deposit DNA. Affymetrix
R
and NimbleGen arrays are produced using a photolithographic process in which