Appl Microbiol Biotechnol (2006) 73:255–273
DOI 10.1007/s00253-006-0584-2
MINI-REVIEW
DNA microarray technology for the microbiologist:
an overview
Armin Ehrenreich
Received: 11 July 2006 / Revised: 11 July 2006 / Accepted: 11 July 2006 / Published online: 17 October 2006
# Springer-Verlag 2006
Abstract DNA microarrays have found widespread use as
a flexible tool to investigate bacterial metabolism. Their
main advantage is the comprehensive data they produce on
the transcriptional response of the whole genome to an
environmental or genetic stimulus. This allows the micro-
biologist to monitor metabolism and to define stimulons
and regulons. Other fields of application are the identifica-
tion of microorganisms or the comparison of genomes. The
importance of this technology increases with the number of
sequenced genomes and the falling prices for equipment
and oligonucleotides. Knowledge of DNA microarrays is of
rising relevance for many areas in microbiological research.
Much literature has been published on various specific
aspects of this technique that can be daunting to the casual
user and beginner. This article offers a comprehensive
outline of microarray technology for transcription analysis
in microbiology. It shortly discusses the types of DNA
microarrays available, the printing of custom arrays,
common labeling strategies for targets, hybridization,
scanning, normalization, and clustering of expression data.
Introduction
DNA microarrays are a powerful tool for the investigation
of various aspects of prokaryotic biology because they
allow the simultaneous monitoring of the expression of all
genes in any bacterium. They offer a more holistic
approach to study cellular physiology and therefore
complement the traditional “gene-by-gene” approaches
A. Ehrenreich (*)
Institute of Microbiology and Genetics, Georg August University,
37077 Göttingen, Germany
e-mail: aehrenr@gwdg.de
(Wildsmith and Elcock 2001). Because the term DNA
microarray was coined in publications from the laboratory
of DeRisi et al. (1996) and Schena et al. (1995), this
technique evolved from a very specialized method that is
only available to few people (Bowtell 1999; Cheung et al.
1999; DeRisi et al. 1997; Lashkari et al. 1997) to a common
tool with many different applications that became important
in microbiology (Dharmadi and Gonzalez 2004). There are
several other types of microarrays, like protein microarrays,
but the DNA microarray is by far the most widespread and
will simply be termed microarray in this review.
The essence of microarray technology is the parallel
hybridization of a mixture of labeled nucleic acids called
target, with thousands of individual nucleic acid species
called probes, that can be identified by their spatial position
in a single experiment. The location of a specific probe on
the array is termed spot or feature. Whereas the probes are
immobilized on a solid support, the targets are applied as a
solution onto the array for hybridization after fluorescent
labeling (Brown and Botstein 1999). The nomenclature of
probes and targets sometimes got mixed up in literature,
but the definition used in this review was given in a
special issue of nature genetics (Phimister 1999) and is
now commonly agreed upon. Transcription analysis with
microarrays is a complex process. Figure 1 gives a brief
overview on the steps involved and discussed in this
review. Much literature has been published on specific
aspects, but the complexity of the topic and the amount of
literature are sometimes daunting to the people starting to
approach the topic. This review wants to give an overview
and explain the major applications of DNA microarrays in
microbiology. It tries to clarify important points, but will
not go into special experimental approaches, details of
equipment, or advanced statistical methods that are not
commonly used in microbiology.
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a Image analysis: Placement
sample 1
extract total
RNA
annotated genomic structure
PCR amplify
probes label RNA using
fluorescent dyes
hybridize labeled
spotting the PCR targets
products
Fig. 1 Main steps in transcription analysis with microarrays. a Probes are
generated from an annotated genome sequence and spotted on a
microarray slide. For target preparation, RNA is extracted from two
experimental conditions and labeled with fluorescent dyes by reverse
transcription. The labeled target is then hybridized with the array, and the
Types of DNA microarrays
Microarrays evolved from Southern blots (Southern 1975,
2001), colony filters (Nguyen et al. 1995), to dot blots
(Kafatos et al. 1979). “DNA macroarrays” or “filterarrays”
were made in a next step of miniaturization by using
robotic devices for spotting thousands of probes on a nylon
membrane. This number was already enough to probe each
gene of a bacterial genome. The targets were labeled
radioactively (Granjeaud et al. 1999), thereby allowing only
one hybridization at a time. The disadvantage of so-called
one-channel experiments is that the variance of each single
array is affecting the final expression ratios. This problem
was solved by two-channel experiments in which two
mRNA populations are labeled with different fluorescent
dyes and are hybridized simultaneously on one array. Glass
slides are commonly used as support because of their low
background fluorescence (Schena et al. 1995, 1996).
Moreover, the rigid glass slides allow much higher probe
density than the flexible membranes of macroarrays,
thereby reducing the amount of target required. Addition-
ally, glass allows covalent linkage of DNA to the surface
and is inert to high ionic strength washing and high
temperature.
There are three main types of DNA microarrays in
widespread use: (1) microarrays where the probes are
Appl Microbiol Biotechnol (2006) 73:255–273
b
of feature indicators and
quantification of flourescence
data
sample 2 image analysis
Background correction and
quality filtering
Data transformation
raw images of
each channel
Data normalization
scan flourescence
signal
Testing for differential gene
expression
Cluster analysis or
biological interpretation
Data storage in local
or public databases
fluorescence of the features is determined using an array scanner. b After
image analysis, quality filtering, data transformation, and normalization
are done. The remaining steps are dependent on the experiment, but in
most cases, the data are tested for different gene expression, clustered,
and finally stored in a database
synthesized in situ directly onto the surface of the chip.
The further two types have in common that independent
synthesized probes are printed on special glass slides.
According to the nature of the probe, they can be classified
as (2) double-stranded DNA microarrays and (3) oligonu-
cleotide DNA microarrays.
Affymetrix GeneChips The most prominent microarrays
with in situ synthesized probes are the GeneChips manu-
factured by Affymetrix (Santa Clara, CA, USA). They are
produced by chemical synthesis of the oligonucleotides
directly on the coated quartz surface of the array (Hughes et
al. 2001; Lipshutz et al. 1999; Lockhart et al. 1996;
Warrington et al. 2000). This technology allows very high
feature densities. It is typical to have 400,000 features on a
commercial array (Lander 1999; Ramsay 1998). Therefore,
they are called high-density oligonucleotide arrays. Gene-
Chips are produced in a unique photolithographic process
analogous to the methods used for production of micro-
electronics chips in combination with chemical reactions
developed for combinatorial chemistry (Fodor et al. 1991).
A quartz wafer is coated with a narrow layer of a light-
sensitive compound. This coating prevents the covalent
coupling of an activated nucleotide. Exposure to light
causes the removal of the chemical protection groups from
the surface. Subsequently applied reactive derivates of
Appl Microbiol Biotechnol (2006) 73:255–273
single nucleotides can then be coupled. The attached
nucleotides again carry a light-sensitive protection group
that has to be removed by illumination before coupling the
next nucleotide. Lithographic masks are used to block or
transmit light onto specific features, thereby determining
the order of nucleotide to be coupled to the growing
oligonucleotides. In repeated cycles of masking, light
exposure, and coupling, oligonucleotides of 25 residues'
length are synthesized on the chip surface. As the
specificity of a probe of 25 nucleotides may not be high
enough, each probe (“match”) is accompanied by a
negative control with a single differing base in the middle
of the probe termed mismatch probe. Performance of probe
and mismatch probe can therefore be used to detect and
eliminate cross-hybridization. Probe and mismatch probe
are called a probe pair. Usually, 11 to 15 probe pairs, called
a probe set, are used to represent a single gene. The very
high feature density in this type of microarray enables the
high number of controls. The automatic production process
guarantees a very high reproducibility and enables a distinct
experimental design: Whereas the DNA microarray types
discussed later in this text are typically used with two
differentially labeled targets, Affymetrix chips are hybrid-
ized with only one labeled target. This allows different
labeling techniques, excludes all dye effects described later,
and eases experimental design and statistical analysis.
However, all those advantages have to be balanced with
the high costs for the design and use of such arrays. A large
number of lithographic masks have to be created, and chip
production is only possible by Affymetrix. This fact almost
excludes any changes to the probes used in a microarray
due to updates of sequences or annotation. Affymetrix
chips are only available for very few microorganisms.
Saccharomyces cerevisiae, Escherichia coli, Bacillus sub-
tilis, Pseudomonas aeruginosa, and Salmonella typhimu-
rium are the only ones listed on the Affymetrix Web site.
Others would require an expensive custom design by the
company. Another fact that has to be kept in mind is that
the whole equipment for hybridizing, scanning, and
analyzing Affymetrix chips are proprietary.
There have been other reports on DNA microarrays
where the probes are synthesized directly onto the chip
surface by using inkjet technology and conventional solid-
phase phosphoamidit technology. However, so far, there is
no widespread use of this technology, although companies
like Agilent Technologies (Palo Alto, CA, USA) are now
using the principle for custom-made arrays (Hughes et al.
2001).
Printed microarrays Here the probes are synthesized
independently and then spotted on the surface of the array
by a microarray spotter (Hegde et al. 2000). There are two
different technologies: contact printer and noncontact
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printer. Contact printers spot the features by various types
of pins. These include split or channeled pins, flat-tipped
pins, and “pin and ring” type of pins (Zhou and Thompson
2004). All the pins initially dip into a solution of the probe
and then onto the slide surface, thereby placing small
droplets in the range of less than 1 up to a few nanoliters on
the surface of the slide. This results in features of 100–
150 μm in diameter, with their centers positioned in a 190-
to 250-μm grid. Before spotting the next probe, the pins are
automatically washed. From one up to hundreds of pins can
be assembled in so-called printheads. The features printed
by one pen of a printhead are sometimes referred to as pen
group or subgrid. Some practical points have to be kept in
mind: when the printhead has multiple pins, their length has
to be perfectly aligned to produce features of similar size.
Even slight misalignments can result in features of varying
size or missing features. There is also a finite number of
features a pin can print before it has to be replaced. It is
very important to confirm by preliminary tests that all pens
produce features of identical size to avoid systematic biases
in the data produced (Hessner et al. 2004).
Noncontact printers use bubble jet (Okamoto et al. 2000)
or inkjet (Lemmo et al. 1998) technology analogous to
computer printers. They shoot small droplets containing the
probe on the surface of the chip. Problems that occur with
this technology are cross-contamination and clogging of the
capillaries, which result in missing spots.
The main advantage of printed microarrays is their
standardized dimension. Historically, the first microarrays
were printed on microscope slides; therefore, a size of
25.25×75.75 mm and a thickness of 1.0 to 1.2 mm is
commonly used. This allows a free choice of spotters,
hybridization equipment, scanners, and software from
different suppliers or to modify slide chemistry. On the
other hand, printed microarrays have a much lower feature
density that can be obtained as compared with GeneChips:
about 10,000 to 30,000 features can be spotted on a single
chip. Although there are prespotted slides for various model
organisms and companies that do commercial custom DNA
microarray spotting, it is also possible to spot the DNA
microarrays in the laboratory (Bowtell 1999; Cheung et al.
1999). However, this is not a cheap undertaking. It is
advisable to install the spotter in a room with a controlled
environment or, better yet, a clean room with regulated
temperature and humidity. This is important because the
small volumes of liquid evaporate quickly, and it is hard to
get reproducible results otherwise. Moreover, dust particles
can interfere with spotting. Because of the costs associated
and the special expertise needed for printing DNA micro-
arrays, many research institutes have microarray core
facilities to handle this task (Searles 2003). Despite these
difficulties, custom-made DNA microarrays offer the
advantage of producing arrays for any species or strains,
258
irrespective of commercial interests. Moreover, it is
possible to print varying numbers of arrays, change slide
chemistry, quickly adjust to progress in annotation, exclude
probes for genes of no interest, or include probes of specific
relevance such as intergenic regions.
Double-stranded DNA microarrays There are two major
types of probes that are used with DNA microarray printers:
double-stranded DNA and oligonucleotides. Double-stranded
DNA commonly results from polymerase chain reaction
(PCR) amplification (Duggan et al. 1999). A 200- to 800-bp
length of amplified DNA is recommended, but larger
fragments of up to 1.3-kb length also work (Heller et al.
1997). In typical microarray design, each probe DNA
corresponds to one gene. This represents the original type
of DNA microarrays where cDNA molecules from Arabi-
dopsis thaliana were amplified by PCR and spotted
(Schena et al. 1995). In prokaryotes, two specific primers
together with chromosomal DNA as template are used to
amplify genes or parts thereof. However, there are also
numerous variations to this strategy. For example, clones
from a shotgun library that originates from a sequencing
project can be used as a template, thereby permitting the
usage of shorter primers. Such clones allow the amplifica-
tion of parts of the genes with only one specific primer.
This way, it is possible to amplify about 80% of the genes
a
- - - - - -
electrostatic interaction
- -
+ + + + + +
NH3 NH3 NH3 NH3 NH3 NH3
NH2
Nucleophilic addition
O H
C HO
Fig. 2 Modes of probe immobilization on microarray slides. a Im-
mobilization of double-stranded DNA on a slide coated with amino-
silane. The negatively charged phosphate backbone is attached to the
positively charged slide surface by electrostatic interaction. Additional
Appl Microbiol Biotechnol (2006) 73:255–273
of a typical prokaryote with only one specific primer,
thereby greatly reducing the costs. Moreover, it increases
the accuracy because only the correct combination of
primers and template results in a PCR product of the
expected size. Nevertheless, the generation of whole-
genome DNA microarrays by high-throughput PCR ampli-
fication is a very laborious and logistically demanding
process. Extensive quality control by gel electrophoresis,
purification of products, and repetition of dropout reactions
is necessary.
The double-stranded DNA is printed on slides with
positively charged coating (Aboytes et al. 2003). In most
cases, they are coated with poly-L-lysine or 3-aminopropyl-
trimethoxysilane (APS). Spotting is typically done with a
1:1 solution of purified PCR products at a final concentra-
tion of 0.2 to 1 mg/ml and dimethylsulfoxide (DMSO)
(Hegde et al. 2000). The DNA is bound to the slide surface
by electrostatic interaction with the negatively charged
phosphate backbone of the nucleic acid as shown in Fig. 2a
(Sanchez-Cortes et al. 2002). This also helps to separate the
two strands of the double-stranded DNA. Additional baking
at approximately 80°C or UV cross-linking is thought to
introduce covalent links primarily of thymine residues in
the DNA to the amino groups of the slide surface (Reed and
Mann 1985; Saito et al. 1981). An additional blocking step
is required to prevent nonspecific interaction of the slide
Covalent coupling
by UV or heat
- - - -
+ + -+ - + - + - -
NH3 NH3 NH3 NH3 NH3 NH3
H2O
Formation of Schiff base
N H N H
C H H C H
covalent linkage is achieved by backing or UV irradiation. b Covalent
attachment of oligonucleotides with a 5′ amino linker to a slide surface
that exposes aldehyde groups
Appl Microbiol Biotechnol (2006) 73:255–273
surface with target DNA especially for arrays printed on
poly-L-lysine coated slides. This “postprocessing” is done
by incubating the slides in a freshly prepared succinic
anhydride solution that readily reacts with the amino groups
from poly-L-lysine (Xiang and Brownstein 2003). The
coated slides are commercially available from many
suppliers but can also be prepared in the laboratory by
intense cleaning of special microscopic slides and dipping
them in poly-L-lysine solution. An advantage of DNA
microarrays made from spotting double-stranded DNA is
their higher hybridization specificity, sensitivity, and their
lower cost. They are indispensable whenever the sequence
of the organism under study is not available. Their biggest
drawbacks are the laborious production of PCR products
and the errors in probe identity that result from mistakes
during their generation. It has been reported that 1 up to 5%
of probes might have a wrong identity in commercial
cDNA microarrays (Knight 2001).
Oligonucleotide DNA microarrays Using synthetic oligo-
nucleotides as probes is an alternative to double-stranded
DNA (Kane et al. 2000; Southern et al. 1999) because they
need much less logistics and are less error-prone due to
automatic manufacturing of the oligonucleotides by the
suppliers and their well-documented delivery in microtiter
plates. Their initial disadvantage of lower specificity and
sensitivity as a result of short oligonucleotides of 25-bp
length have been overcome by using longer probes with a
length of 50 to 70 bp (Barczak et al. 2003; Bates et al.
2005; Calevro et al. 2004). This short probe length is a
major advantage of oligoprobes because it allows the
monitoring of the transcription of very small open reading
frames or to focus transcription analysis to intergenic
regions. However, oligonucleotides as probes require a
careful design (Emrich et al. 2003; Herold and Rasooly
2003; Rouillard et al. 2003). All calculated melting points
must fall into a temperature range of 5°C, and self-
homology has to be avoided. Because of their short size,
oligonucleotides are commonly attached to the slide surface
by covalent coupling. Otherwise, a significant amount of
probe would be lost from the array surface during
hybridization and washing. A large multiplicity of chemical
reactions has been proposed to achieve covalent coupling,
but the majority of slides used for spotting oligonucleotides
are coated with compounds providing aldehyde or epoxy
functional groups. To achieve covalent linkage, oligonu-
cleotides with modifications at the 5′ or at the 3′ end are
used. This increases the availability of the probe sequences
for hybridization with target because it is not fixed to the
surface by its backbone or bases. A further increase in
sensitivity can be obtained by inserting spacer molecules
between the oligonucleotide and the slide surface (Beier
and Hoheisel 1999; Ghosh and Musso 1987; Shchepinov et
259
al. 1997). The most common modification of oligonucleo-
tides is a 5′ amino group (Zammatteo et al. 2000). It offers a
high flexibility in the choice of slide chemistry: as
illustrated in Fig. 2b, aldehyde and epoxy groups react
especially readily with the primary amino group. Modified
oligonucleotides are normally spotted at a concentration of
10–30 μM. The conditions have to be adjusted so that the
coupling can proceed. Finally, the functional groups of the
array that are not part of a feature have to be blocked,
similar to arrays made from double-stranded DNA probes.
Depending on slide chemistry, this can be done, for
example, by incubating the slides in the presence of low
molecular primary amines. Printed microarrays can be
stored for many months if they are protected from light
and kept under completely dry conditions in a desiccator
(Worley et al. 2000).
Methods used for target labeling
Many different fluorescent dyes and other labeling agents
have been described in the literature (Badiee et al. 2003;
Schena and Davis 2000), but the cyanine dyes Cy-3 and
Cy-5 are most commonly used, offering strong fluores-
cence, similar chemical properties, well-separated fluores-
cence spectra, and little adherence to chip surface.
In contrast with common expectation, they are not green
and red themselves, but they get those colors only after
scanning by computer false coloring. There are two main
strategies for their incorporation in cDNA by reverse
transcription (RT) of RNA (Wildsmith et al. 2001).
Direct labeling In the direct labeling protocol, the dye is a
derivative of a nucleotide triphosphate, like Cy-3 deoxyur-
idine triphosphate (dUTP) or Cy-3 deoxycytosine triphos-
phate (dCTP). It is incorporated during RT of the RNA into
cDNA. One of the deoxynucleoside triphosphates (dNTPs),
either the dCTP or the dUTP, needed by the reverse
transcriptase is provided at lower concentration. In addition,
the derivative of the corresponding dye is added (Khodursky
et al. 2003), resulting in incorporation of the dye. For two-
channel experiments, RNA prepared from cells grown at
two different conditions is included in the hybridization
experiment. One of the RNA preparations is labeled with
Cy-3, the other with Cy-5. After labeling and removal of
remaining free dye, roughly equal amounts of Cy-3 and
Cy-5 dye incorporated in the cDNA are subjected to hy-
bridization. Whereas direct labeling is more widespread, it
has the fundamental problem that Cy-3 and Cy-5 are
incorporated with different yields. In practice, this differ-
ence can be quite substantial because the Cy-3 and Cy-5
molecules have a different size. This results in a lower rate
260
of integration of the Cy-5-modified nucleotide in cDNA as
compared with the Cy-3-modified one. This artificial bias
has to be corrected by normalization to obtain relevant
biological data.
Indirect labeling To circumvent this major source of error, a
different strategy of labeling called indirect labeling is used.
In this case, both RNA preparations are reverse-transcribed
to cDNA in the presence of an aminoallyl-modified
dUTP or dCTP, respectively. Since both preparations are
labeled with the same molecule, there is no bias.
Additionally, this modification is much smaller than Cy dyes
and, thus, better incorporated in the cDNA. In a second step,
N-hydroxysuccinylimidyl ester (NHS ester) derivatives of
Cy-3 or Cy-5 are coupled to the aminoallyl-modified cDNA
molecules by a chemical reaction that is far less sensitive to
the molecule size of the dye. The disadvantages of this
protocol are the extreme moisture sensitivity of the NHS
ester–modified dyes and the requirement of significantly
more bench work. The often-stated advantage of requiring
less RNA as starting material is neutralized by the losses due
to the two purification steps.
Labeling of genomic DNA Labeled genomic DNA is used
for comparative genomic studies (Borucki et al. 2003; Chan
et al. 2003; Salama et al. 2000) as a reference target in
normalization or for slide quality control. The labeling is
usually done by direct incorporation of Cy-3- or Cy-5-
labeled nucleotides in a nick translation or by random
priming with the Klenow fragment of DNA polymerase.
For the random priming, DNA fragments of 1- to 3-kb sizes
are usually generated by sonication, nebulization, or by
digestion with restriction enzymes with a four-base recog-
nition site, like AluI or Sau3AI. For a single hybridization,
a labeling reaction contains 0.5–2 μg of genomic DNA
(Amon and Ivanov 2003; Ye et al. 2001).
Target preparation A specific problem of working with
prokaryotic organisms is that there are no widely adopted
protocols for selectively labeling the mRNA. Whereas the
mRNA of eukaryotic organisms has a poly(A) tail that can
be utilized to specifically label mRNA with oligo(dT)
primers, prokaryotic mRNA lacks the poly(A) tails, and
random priming either with hexamers or nonamers has to be
used. Therefore, only total RNA can be labeled. But only 4%
of the total RNA is mRNA, the rest being mainly rRNA and
tRNA (Neidhard et al. 1990; Talaat et al. 2000). The large
amount of labeled RNA results in a higher background in
DNA microarray experiments with prokaryotic organisms
and requires a substantial higher amount of total RNA to be
added to the labeling reaction. Although lower numbers
have been published for certain protocols, as a rule of
thumb, 20 to 25 μg of total RNA have to be included in a
Appl Microbiol Biotechnol (2006) 73:255–273
labeling reaction of a prokaryotic organism whereas only 2
to 5 μg of total RNA are needed for a eukaryote (Duggan et
al. 1999). Numerous attempts to circumvent this problem
have been published such as preparation of polyadenylated
mRNA from prokaryotes (Wendisch et al. 2001) or priming
with a primer set that has a higher probability of priming
the RT of mRNA than of rRNA (Talaat et al. 2000).
However, none of them has been widely adopted. An
additional major problem when working with prokaryotic
mRNA as compared with eukaryotic mRNA is its distinct
instability. Prokaryotic mRNA only has a half-life in the
range of 40 s to 20 min for individual transcripts (Kushner
1996). The average, as measured with isotopic labeling, is
around 1 min (Baracchini and Bremer 1987; Neidhard et al.
1990), whereas microarray experiments indicate that 80%
of E. coli transcripts have half-lives ranging from 3 to
8 min (Bernstein et al. 2002). Because bacterial RNAses are
responsible for this rapid turnover (Kushner 2002), this
clearly indicates that prokaryotic mRNA is much harder to
handle than eukaryotic mRNA. This instability demands
special care during preparation of prokaryotic RNA to
avoid artifacts. It is possible to fail to observe the
expression of certain genes simply because of degradation
of the corresponding mRNA during the experiment. It also
has to be kept in mind that depending on the promotor and
its regulation in E. coli transcription initiation takes place
at a rate of once per second to once per generation (Record
et al. 1996). The transcript elongation proceeds at a rate of
40–50 nucleotides per second (Richardson and Greenblatt
1996). This means that even for a relatively large protein,
like lacZ, the first β-galactosidase proteins appear 1 min
after the initial signal for gene induction occurred. This
should illustrate how quickly microorganisms adjust their
transcription to environmental changes that might occur
during harvesting the culture and cell disruption. Therefore,
to prevent observing mainly the Save Our Souls (SOS)
response or the response to oxygen limitation, it is critical
to immediately cool cells carefully during harvest and use
an appropriate method for cell disruption and RNA
extraction that minimizes RNA degradation. Traditional
methods of cell disruption, like incubation with lysozyme,
french pressing, or sonification, are often not suitable
because they take too much time. A very flexible solution
that works for many organisms is to freeze cells using
liquid nitrogen and grind the frozen cells in a cooled ball
mill. The resulting powder of grounded cells is dissolved in
a buffer containing a high concentration of the strong pro-
tein denaturant guanidinium isothiocyanate before thawing,
thereby inhibiting any RNAse activity. Alternatively, a cold
“stop solution” composed, for example, of ethanol and
phenol at a low pH can be used to stop any transcription
immediately and prevent RNA breakdown (Moore et al.
2005). Total bacterial RNA can then be prepared with
Appl Microbiol Biotechnol (2006) 73:255–273
commercial kits such as RNeasy from Qiagen (Hilden,
Germany). The quality of RNA is pivotal for transcription
analysis, and RNA quality should be controlled, for
example, by denaturing formaldehyde agarose gel electro-
phoresis or RT PCR.
Hybridization and data acquisition
Hybridization Hybridization of DNA microarrays can be
done in two different ways. The “classical” approach
includes placing labeled, denatured target on a slide and
carefully covering it with a coverslip. This requires some
skillfulness because the coverslip needs to be level to
prevent gradients in hybridization and avoid trapped air
bubbles. The slide is then placed in a humid chamber to
prevent desiccation during hybridization and incubated at
the hybridization temperature. The hybridization tempera-
ture ranges mostly from 40 to 65°C for 5 to 12 h. It depends
on the organism studied and the composition of the
hybridization buffer (Cheung et al. 1999). In most cases,
saline sodium citrate (SSC) buffer with added detergent is
used. Addition of Denhardt's solution, sheared salmon
sperm DNA, or tRNA reduces the background. The
addition of formamide, dextran sulfate, or polyethylene
glycol can improve binding of low-copy number transcripts
(Cheung et al. 1999; Wildsmith and Elcock 2001).
Hybridization temperature is critical for oligonucleotide
slides and has to be carefully optimized. As a rule of
thumb, the optimization can start at a hybridization
temperature 15°C below the mean melting temperature of
the oligonucleotides used. Following hybridization, the
slides are washed to remove unspecific bound target. More
stringent washing steps are performed at the end of the
washing procedure. This can be achieved either by
decreasing the ionic strength or increasing the washing
temperature (Wildsmith and Elcock 2001). Typical proto-
cols use decreasing SSC buffer concentrations first with
small concentrations of sodium dodecyl sulfate (SDS) then
without SDS. The slides are finally dried by centrifugation.
It is important to scan the arrays within several hours after
hybridization because the fluorescence signal deteriorates
with time.
As an alternative to this classical approach, automatic
array hybridization stations can be used (Wildsmith and
Elcock 2001). They provide hassle-free hybridization and
washing of the slides by running programmed protocols.
The results do not depend on the ability of the researcher
and are very reproducible. However, hybridization and
washing conditions have to be fine-tuned in earlier experi-
ments to the probes and slide chemistry used. They are
therefore most adequate when a large number of arrays
261
based on the same chemistry have to be handled identically.
They are not well suited to deal with small number arrays
based on varying chemistries.
Scanning The microarrays are scanned with microarray
scanners. Their appropriate driver and image analysis
software determines the raw values (Bassett et al. 1999).
GenePix (Axon Instruments, Inc., Union City, CA, USA)
and ArrayVision (Imaging Research, Ontario, Canada)
software are examples of widely used softwares for image
analysis and raw data acquisition. In principle, it is possible
to scan standard-sized slides with any scanner. Exceptions
are a few slide types with a nonplanar surface that exclude
confocal scanners. For successful data acquisition, a data
file is needed that identifies the features and defines their
dimensions and locations. The GenePix array list (.gal) file
format is often used for this purpose. The scanners mostly
use lasers for exciting the surface of the hybridized
microarray with a resolution of a few micrometers (Bowtell
1999). The resolution of scanning should be better than
10% of the spot size, that is, features of 150-μm size need
to be scanned at least at 15 μm resolution. The fluorescence
emitted from the dyes hybridized to the features is collected
and quantified by photomultiplier tubes or charge-coupled
device (CCD) cameras. There is a variety of scanners on the
market differing in their technological configurations
(Ramdas et al. 2001). Normally, the scanner generates
gray-scale images of the fluorescence at 532 and 635 nm.
The data are stored in a lossless tagged image file format
(TIFF) that is used for quantification by image analysis. A
color depth of 2 byte is characteristic for most scanners,
which means that each pixel can assume 65,535 different
intensity levels. The sensitivity of the scanner has to be
adjusted to ensure that most of the pixels in the picture do
not saturate its dynamic range. It is convenient to roughly
adjust the sensitivity of the scanner during a prescan so that
constitutive controls result in roughly equal signals. For
microarrays made from double-stranded DNA, this can also
be done by spotting chromosomal DNA and adjusting these
spots to a ratio of 1 during scanning.
Image analysis To quantify the fluorescence of the features
via image analysis, pixels have to be assigned either to a
spot or the background. This resulting boundary is often
visualized in the software for acquiring raw values by a
circle surrounding the feature and is then called a feature
indicator. In most cases, the image analysis software allows
the placement of the feature indicators in a semiautomatic
or automatic manner. Even if an algorithm places the
feature indicators automatically, it is advisable to manually
control this placement. In real life, the spots might be
irregular, or fluorescent impurities on the chip surface may
confuse algorithms. It is common to define all pixels inside
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a feature indicator as foreground and all adjacent pixels
within a radius of three times the feature diameter as the
local background. The next step is the quantification of the
image data by calculating the arithmetic mean (Zhou et al.
2000) or better median (Petrov et al. 2002) of the intensities
of the foreground and background pixels. This resulting
data are stored in form of a table. Common spreadsheet
programs and all sorts of commercial and free software
tools can be used for the next steps of data analysis
(Brazma and Vilo 2001; Conway et al. 2002).
Background correction and filtering First, the so-called
background correction has to be made. That simply means
subtracting the local background value from the foreground
intensity (Benes and Muckenthaler 2003; Dharmadi and
Gonzalez 2004). Additionally, an intensity-based filtering
of the data should be done to ensure the quality of the
signals and to prevent artifacts. The first and most
important of these quality assessments is to exclude features
with intensity smaller than the background or assign them a
“floor” value, which is often the local or global back-
ground. The assignment of a floor value allows interpreta-
tion of genes that are transcribed at one condition but are
totally switched off at the other condition. This is not
uncommon with bacteria where some operons are specifi-
cally induced by an inducer but are not transcribed in its
absence. Because the data at the lower range of intensities
tend to be much more variable, it is good practice to accept
only the intensity of features that lie significantly above
background and assign floor values to the rest. Significantly
above background means that they should have intensities
that are more than one or two standard deviations above the
local background. Two standard deviations above back-
ground mean that they represent valid data with a
confidence level of 95.5% (Quackenbush 2002). After
passing this filtering, the ratio of means or the ratio of
medians is calculated from the background-corrected
intensities in the “red” and “green” channels and results in
the actual raw data from a DNA microarray experiment. A
further filtering that can be applied to verify quality and
prevent experimental artifacts would be the comparison of
the ratio of means, the ratio of medians, and the regression
ratio for each feature. The regression ratio is the linear
regression between the intensities of pixels within a circle
of twice the diameter of the feature. The slope of the line of
best fit according to the least-square method is the
regression ratio. The distinct feature of this ratio is its
independence of rigidly defining the background or
foreground pixels. Whenever the regression ratio, the ratio
of means, and the ratio of medians deviate too much from
each other, there is a problem with spot morphology or
feature indicator placement. These data should be omitted
from further analysis. For example, the GenePix software
Appl Microbiol Biotechnol (2006) 73:255–273
exports all values needed for the quality assessments
described.
Data transformation The untransformed values of expres-
sion ratios have the disadvantage of treating up- and
downregulated genes differently. That means that a fourfold
upregulated gene has an expression ratio of 4, whereas a
fourfold downregulated gene has an expression ratio of
0.25. To circumvent this problem, the expression ratios are
often handled as their logarithms to the base 2. This results
in the values 2 and (−2) for a fourfold up- and down-
regulated gene, which has a number of practical advan-
tages. There is another important reason for transforming
data to their logarithms: most statistical methods applied on
the data afterward expect normal distribution. Log-trans-
forming data is a mathematically simple strategy to achieve
this (Dharmadi and Gonzalez 2004). Figure 3 shows two
common types of plots that are used to visualize microarray
data.
Methods for normalization and testing for differential
gene expression
Normalization Before it is possible to draw biological
conclusions or to apply sophisticated statistics, it is
important to normalize the data. This corrects for systematic
biases resulting basically from different amounts of RNA
used for labeling, different incorporation efficiencies of the
Cy-3 and Cy-5 dyes in the labeling protocols, and different
detection efficiencies of the dyes (Yang et al. 2002a,b). A
number of normalization methods have been proposed in
the literature (Duggan et al. 1999; Kroll and Wolfl 2002;
Quackenbush 2002). The most widely used method is
based on the assumption that the total sum of intensities
should be equal in both channels, and therefore, the ratio
between them should be one. A normalization factor is
calculated from overall ratio, and ratios for all features are
scaled accordingly. A somehow similar approach uses
spotted chromosomal DNA for normalization, although this
strategy is confined to microarrays made by spotting
double-stranded DNA (DeRisi et al. 1997). In addition, a
number of other approaches are in use such as linear
regression analysis and the intensity-dependent locally
weighted linear regression (LOWESS) normalization that
corrects for intensity-dependent effects sometimes observed
in the data. A microbiologist has to be cautious with these
normalization strategies because they imply statistically that
there must be a downregulated gene for every upregulated
one. This assumption might be more appropriate from a
statistical point of view with large eukaryotic genomes. It is
not necessarily correct for bacteria because the number of
Appl Microbiol Biotechnol (2006) 73:255–273
a b
4
3 1
log2(R/G)
log10G
2
1
1 2 3 4
log10R
Fig. 3 Plots used to represent microarray experiments. a In a scatter
plot, each feature represents a point. The coordinates are the logarithm
of the intensities in the red and green channel. In this plot, normalized
data should map around the bisecting line. b In an R-I plot, the
genes is much smaller. Bacteria can regulate large groups of
genes in a highly coordinated fashion, and the total amount
of mRNA may be vastly different. An extreme example that
is nevertheless common in microbiological research is the
comparison of cells with a significantly different growth
rate. In such cases, it is better to normalize by using
“housekeeping genes”. This means that the normalization
factor is calculated from genes that do not change their
expression level under the given experimental conditions
(DeRisi et al. 1996). Of course, one has to be careful in
selecting those genes especially with “nonstandard” organ-
isms because this method requires some preceding exper-
imental experience with gene expression in this organism.
The normalization factor is then calculated from a small set
of genes fulfilling this condition. If such knowledge is not
available, an alternative for normalization is by using
external controls. This strategy is similar to the use of
housekeeping genes but does not correct for different
amounts of RNA in the target preparations. Therefore, it
is best combined with another normalization strategy.
Exogenous controls require spotting of control genes on
the array that have no homology to genes of the genome
under study and, therefore, will not exhibit cross-hybrid-
ization. These controls need to have similar general
characteristics, that is, melting temperature, guanine–cyto-
sine (GC) content, and length compared with the rest of the
probes on the chip. RNA complementary to those genes is
263
3
2
-1
-2
-3
log10(R*G)
logarithm of the ratio of the red and green intensity for each feature is
plotted as a function of the product of intensities. This plot easily
reveals systematic intensity-dependent bias
transcribed in vitro and added to both labeling mixes in
known concentrations and ratios. These ratios can then be
used to calculate a normalization factor. The detection limit
and the saturating concentration of the experiment can be
estimated from a row of increasing concentrations of
controls. External controls with no complementary spiked-
in RNA can be used as negative controls to judge the
stringency of the washing protocol.
Testing for differential gene expression The following steps
of transcription analysis highly depend on the question the
experiment addresses. Nevertheless, it is of interest for most
experiments performed which genes are differentially
expressed. Although many strategies have been proposed,
the most widely used is to set a fixed fold-change cut off
(Cui and Churchill 2003; Yang et al. 2002a,b). Usual values
are two- to fivefold because DNA microarrays have been
shown to be reproducible at this level, especially when the
data are validated by repeated experiments (DeRisi et al.
1996). This approach seems straightforward and performs
well especially with the relative strong gene regulation in
microorganisms. To detect more subtle regulations, it is
better to calculate the mean and standard deviation for the
specific data set. Afterward, every data point can be
transformed to its Z score (Quackenbush 2002). This
number simply describes how many standard deviations it
is above or below the mean. By defining the cut off in
264
terms of a Z score, the threshold is more specific to the
particular data set. DNA microarray data tend to be more
variable at lower than at higher intensity levels. Therefore,
it is better to define an intensity-dependent Z score
threshold for the data set. For this, the mean and standard
deviation are calculated in a sliding window to establish a
local intensity-dependent Z score for each data point (Yang
et al. 2002a,b). Values with an associated Z score larger
than 1.96 can be regarded as differentially expressed genes
at a 95% confidence level (Quackenbush 2002).
Design of experiments and data verification
Replication Replication is crucial to achieve reliable data
for microarrays (Spruill et al. 2002). There are three
different kinds of replication that have to be distinguished.
Spotting the same probes multiple times on each array is
the first one. This provides some backup in case a spot
cannot be evaluated due to technical artifacts, like dye
precipitations or dust particles, and allows the calculation of
the “on chip variance” (Worley et al. 2000). Moreover, it
improves the data quality by calculating the average
expression ratios on the chip. The next important type of
replication is to label and hybridize RNA that has been
prepared from one biological experiment several times.
This corrects variance that results from differences in the
labeling reactions. The most important replication in this
category is the so-called dye switch or dye swap. In a dye
switch, the RNA sample that was first Cy-3-labeled is
Cy-5-labeled next and vice versa (Kerr and Churchill
2001a,b; Tseng et al. 2001; Yang and Speed 2002). This
is important because there are gene-specific dye effects.
Additionally, the intensity of Cy-3 and Cy-5 fluorescence is
differing depending on the amount of dye that is bound to a
feature by hybridization (Rhodius et al. 2002).
All replications mentioned so far are often called
technical replications. They are only used to correct
technical sources of error during the transcription analysis.
An additional “technical” source of variability is the RNA
preparation. However, it should be kept in mind that the
major varieties come from the biological experiment that
has to be planned and conducted with great care. Micro-
arrays monitor the mRNA concentration of all genes of a
cell with considerable precision. Bacteria in turn can detect
environmental changes with extreme sensitivity, and
mRNA concentrations show a rapid change in response to
them. Therefore, the major task when trying to get
reproducible microarrays is to perform highly reproducible
physiological or genetic experiments. Good array data
depend as much upon good microbial physiology technique
as they do on good DNA array technique (Conway and
Schoolnik 2003).
Appl Microbiol Biotechnol (2006) 73:255–273
A statistical estimation deduces that at least three
replicates should be done (Lee et al. 2000). The ratios of
replicate experiments are averaged or better the geometric
mean is calculated (Quackenbush 2002).
Design of experiments The experimental design is also
critical (Churchill 2002; Kerr and Churchill 2001a,b;
Nadon and Shoemaker 2002). For example, many genes
in a bacterial cell are growth-rate-dependent. Therefore, one
has to be very cautious to attribute the gene expression
measured on different growth substrates if the growth rates
of the cultures are too different. A possibility to circumvent
this problem is to work with cells grown in continuous
cultures where the growth rates can be adjusted to be
similar. However, this approach excludes the observation of
dynamic responses.
The common case in microarray research is to compare
transcription of two cell populations. Three groups of these
two-condition experiments can be generally distinguished
(Conway and Schoolnik 2003): (1) differential response to
growth parameters. Examples for this would be studies of
growing cells either in batch or in continuous culture on
various carbon sources or other varying growth conditions
such as aerobic vs fermentative growth (DeRisi et al. 1997;
Oh et al. 2002; Pappas et al. 2004; Pashalidis et al. 2005;
Paustian et al. 2002; Polen et al. 2003, 2005; Rossignol et
al. 2003). (2) Treated vs untreated cultures. These experi-
ments monitor the response of a cell population to various
physiologic challenges such as, for example, heat shock or
antibiotic treatment. They define genes belonging to certain
stimulons in the cells and provide a picture on how the cell
copes with certain stress situations (Alsaker and Papoutsakis
2005; Anthony et al. 2005; Beckering et al. 2002; Chhabra et
al. 2006; Gao et al. 2004; Mascher et al. 2003). (3) Wild-type
vs mutant strains. This group summarizes studies where the
consequences of genetic mutations are monitored. They are
often employed to define regulons, but special care must be
taken to separate direct consequences of the mutation from
indirect ones (Barbosa and Levy 2000; Cao et al. 2003; den
Hengst et al. 2005; Ogura et al. 2002; Salmon et al. 2005).
If more than two conditions are to be investigated, for
example, time rows (Belland et al. 2003; Kucho et al.
2005), a single chip experiment is not enough, and several
hybridizations have to be combined (Yang and Speed
2002). It is important to carefully plan this experiment to
generate meaningful data, detect possible biases, and
avoid that the factors of interest are masked by the adding
errors. Figure 4 shows examples of such experimental
designs. The common reference design, as shown in
Fig. 4a, is the predominant case (Conway and Schoolnik
2003). A problem with using references is that many
genes in bacteria are completely switched off without
their inductor. Therefore, a number of them will therefore
Appl Microbiol Biotechnol (2006) 73:255–273
a genomes and identify microorganisms. These fields of
sample 1 sample 2 sample 3 sample 4
reference
b corresponding probe will yield a signal in both channels,
sample 1
sample 2
sample 5
sample 4 sample 3
c 2005; Reen et al. 2005).
sample 1
sample 5 sample 2
sample 4 sample 3
Fig. 4 Basic types of experimental design schemes with multiple
samples. Each box represents one sample, and each arrow points from
the green-labeled sample to the red-labeled sample. a Reference
design; b loop design; c all-pair design
not be expressed under the condition the reference was
made. If any of those genes are expressed under other
conditions, this will lead to infinite induction ratios for
those genes. Some studies suggest using a mixture of
reference RNAs obtained from several sampling condi-
tions (Kucho et al. 2005; Laub et al. 2000), labeled
oligonucleotides complementary to each probe (Dudley et
al. 2002), or to use labeled chromosomal DNA as
reference (Belland et al. 2003). Another strategy would
be to define a base value in case there is a strong signal
on one channel but no signal for that feature on the other
to be able to calculate a ratio.
Other experimental approaches with microarrays Beside
transcription analysis and many other minor applications,
microarrays are routinely used in microbiology to compare
265
application require the labeling of chromosomal DNA that
is hybridized with the array. For the comparison of
genomes, also called genomotyping, the chromosomal
DNA from the bacterium that has to be typed is labeled.
This labeled DNA is hybridized with a whole genome array
from a reference strain. The chromosomal DNA from the
reference strain is labeled with a second dye and included
in the hybridization. If genes are present in both strains, the
whereas when the gene is absent in the typed strain, the
signal in one channel is missing. Threshold values are
defined on the basis of the data set to decide whether a gene
is absent. This decision is often done with the GACK
software (Kim et al. 2002; Stabler et al. 2005). The
approach works only with closely related organisms and
has the inherent limitation that is only possible to decide
which genes are missing from the typed strain as compared
with the reference strain, not which ones the typed strains
has more than the reference strain (Coenye et al. 2005;
Lindroos et al. 2005; Molenaar et al. 2005; Paustian et al.
For the identification of microorganisms, probes specific
to certain taxons are spotted on arrays. Mostly, 16S and 23S
ribosomal RNA genes are used for this purpose. The labeled
chromosomal DNA of the organism under investigation is
hybridized with this chip in a one-channel experiment. By
sophisticated design of these probes, it is possible to classify
the organism depending on the probes that hybridize with
the target (Belosludtsev et al. 2004; Lehner et al. 2005; Loy
et al. 2002, 2005; Mitterer et al. 2004).
Data verification Microarray data can easily contain errors
originating from probe interchange, array production,
labeling reactions, hybridization, and data acquisition.
Therefore, it is crucially advisable to validate data of the
most important genes with independent methods to quantify
mRNA. Real-time RT-PCR (Gibson et al. 1996; Heid et al.
1996; Helmann et al. 2001; Wurmbach et al. 2003) or
Northern blotting (Heller et al. 1997; Schuchhardt et al.
2000) are the most common options. In prokaryotic
organisms, the operon structure can also give some hints:
operons are expressed as coordinated and often show a
polar effect in the direction of transcription (Pappas et al.
2004). However, more indirect methods, like proteomics
data, lacZ fusions, or enzyme activities, can also be used to
back up transcription data (Rhodius et al. 2002). Moreover,
they proof the relevance of the transcription data for these
levels of cellular physiology. The best option to verify
microarray expression data is real-time RT–PCR, also
called QRT-PCR (quantitative RT-PCR). This PCR method
allows to quantify dozens of samples simultaneously. It has
been shown that in most cases, the data from microarrays
266
and real-time RT-PCR are consistent (Mutch et al. 2002).
However, in the majority of studies, microarray data
compress the fold changes of expression as compared with
real-time RT-PCR by two- to tenfold (Conway and Schoolnik
2003). This has been attributed to the smaller dynamic range
of microarrays (Holland 2002; Pappas et al. 2004; Yuen et al.
2002). Northern blotting is another option for data valida-
tion, but it is only applicable for much fewer sample
numbers and is less quantitative. If a larger number of
samples are to be checked, RNA dot blot analysis has been
used to verify array data (Moore et al. 2005).
Data storage Microarrays produce a vast amount of data. It is
important to organize and store these data in databases (Bassett
et al. 1999; Brazma et al. 2002; Sherlock and Ball 2005). This
is true for the work in the laboratory and for the deposition of
the data in public databases. Many journals require the
deposition of the microarray data in public databases, like
“National Center for Biotechnology Information (NCBI) Gene
Expression Omnibus” (GEO) (Edgar et al. 2002), “KEGG
EXPRESSION” database (Kanehisa et al. 2002), “ArrayEx-
press Database” (Brazma et al. 2003), or “Stanford Microarray
Database” (Ball et al. 2005) and the submission of the
assigned accession number prior to publication (Brazma et al.
2000). The minimum information about a microarray exper-
iment (MIAME) specification was created to achieve accurate
and consistent annotation of microarray experiments (Ball et
al. 2002; Brazma 2001; Brazma et al. 2001). This specifica-
tion tries to define a framework to describe the minimal data
set required for a microarray experiment. It comprise data on
the experimental design, information on the array design, the
samples used, the RNA extraction and labeling, hybridization
procedures and parameters, experimental data, and finally, a
detailed description of strategy and controls used for
normalization. The experimental data comprises image data,
raw data, data after normalization, and after averaging of
replicates. For describing all these data in a structured way, a
special XML format called microarray gene expression
markup language (MAGE-ML) has been proposed (Spellman
et al. 2002). The databases also offer online tools for data
submission, like MIAMExpress (Brazma et al. 2003).
Cluster analysis of expression data
The underlying principle of applying clustering to expres-
sion data is the assumption that similar expression levels
might indicate related biological function (Brazma and Vilo
2001). Therefore, insight in the function of unknown genes
may be gained by observing whether they are coregulated
with known genes or whether the genes are expressed or
repressed as a group in response to a defined stimulus.
Appl Microbiol Biotechnol (2006) 73:255–273
Answers to these questions can be obtained by applying
clustering algorithms (Claverie 1999; de Hoon et al. 2004;
Eisen et al. 1998; Michaels et al. 1998; Sherlock 2000;
Sturn et al. 2002). Typical software packages used for this
step are GeneSpring (Silicon Genetics, San Carlos, CA,
USA) or SpotFire Array Explorer (SpotFire, Inc., Cam-
bridge, MA, USA). The interpretation of transcription data
in the context of known functions, for example, biochem-
ical pathways or a working model, is a hypothesis-based
approach. In contrast to this, a purely statistical analysis,
like clustering, can be employed without any prior
hypothesis and might, at least in theory, lead to unexpected
conclusions. This “unsupervised analysis” is seen as a
major advantage of the functional genomics approach.
However, it has to be stressed that clustering of data will
always result in some clusters regardless of biological
relevance (Clare and King 2002). This is a common
characteristic of bioinformatics data. It might suggest
totally unexpected coherences but does not prove anything
per se without returning to the laboratory and doing
classical experiments to find supporting evidences.
Clustering is applied to the averaged expression data from
several microarray experiments where quality assessment,
normalization, and technical controls have already been
done. It can be applied to several distinct experiments or a
time series. It is possible to cluster genes in groups according
to the similarity of the expression in several experiments or
to cluster the experiments to groups of similar gene
expression. When these two clustering directions are
combined, they are referred to as biclustering or two-way
clustering (Cheng and Church 2000; Getz et al. 2000).
To simply cluster the genes according to their expression
in several experiments, the expression level of each gene is
represented as a point in an n-dimensional space. n is equal
to the number of experiments or time points. This can easily
be visualized with data from two experiments because the
points (genes) can be represented by a two-dimensional
coordinate system as shown in Fig. 5. Clusters can, under
these circumstances, be visually recognized as points in
closer vicinity.
Before any mathematical clusters analysis can be done,
three things need to be defined:
– First, a distance measure between data points has to be
selected. Most often, this is the Euclidian distance
between points, but more sophisticated definitions are
possible and result in different clusters. Therefore, this
can be as important as the selection of the clustering
algorithm. Some sort of “feeling” for the data set and
trial and error is used for the choice of the right one.
– Second, a function that defines the quality of clustering
results must be chosen. Most obviously, the defined
distance measurement is used to minimize the distance
Appl Microbiol Biotechnol (2006) 73:255–273 267

Fig. 5 Schematic illustrating

how clustering algorithms work.
a
In this example, data from only
two experiments result in a two-
dimensional data set. a Hierar-

Experiment 2
chical data clustering produces a
tree structure. b In K-means
clustering, centroids, drawn as
stars, are dispersed by the user,
and data points are assigned to
the clusters in an iterative algo-
rithm. c Self-organizing maps
start with a regular grid of
centroids, represented as stars.
Pulling the centroids to the
centers of the clusters they rep-
resent in an iterative algorithm Experiment 1
b
identifies the clusters

Experiment 2
Experiment 2

Experiment 1 Experiment 1
c
Experiment 2
Experiment 2

Experiment 1 Experiment 1

of each point in a cluster to the center of the cluster.
But again, other methods are possible and necessary,
depending on how noisy the data are.
– Finally, the algorithm for clustering needs to be selected.
These algorithms try to find the best possible clustering
results using the function that defines the quality of
clustering. There is a vast collection of clustering
algorithms described in the literature. The major ones
can be separated into two groups depending on whether
the user needs to make assumptions on the clusters a
priori and specify the number of clusters to be found.
Hierarchical clustering Hierarchical clustering as illustrated
in Fig. 5a is a widely used method that does not need a
priori information and results in a tree structure of
increasing similarity (Khan et al. 1998; Lashkari et al.
1997; Schena et al. 1996). Every tree node represents a
268
cluster at some resolution. The size of the resulting clusters
can therefore be defined by setting a threshold of a certain
intercluster distance. The hierarchical clustering algorithms
work either “top-down,” by starting with all genes in a
single cluster and separating them based on a criterion of
dissimilarity, or “bottom-up,” by starting with every
individual gene in a single cluster and merging them
consecutively based on a criterion of similarity. After
hierarchical clustering, trees and color maps are the most
natural representation of results (Wen et al. 1998).
Hierarchical clustering gives good results with a clean data
set but is very sensitive to noisy data.
The two other clustering algorithms mentioned here
require an initial guess on the number of suspected clusters.
K-means clustering K-means clustering, an alternative to
hierarchical clustering, is argued to give good results when
compact clusters are expected. As shown in Fig. 5b, the user
disperses so-called centroids in the data space. The iterative
algorithm assigns each data point to the cluster with the
smallest distance to the next centroid. It then calculates a
new one from the points that belong to the cluster and
replaces the old centroid. The computation process is
terminated when there is no further change in the assignment
of gene points to the centroids (Lu et al. 2004; Xu 2004).
Self-organizing maps Self-organizing maps (SOMs) are
clustering algorithms that are related to K-means clustering
but have been found to be superior in both robustness and
accuracy when analyzing microarray data (Alsaker and
Papoutsakis 2005; Garge et al. 2005; Tamayo et al. 1999).
The algorithm, illustrated in Fig. 5c, is complex and starts
with centroids dispersed as a regular grid among the data.
Each data point then pulls the nearest centroid in little steps.
The extent of pulling depends on the distance to the
centroid. Centroids that come close to each other merge,
and centroids with no movement will be deleted. When the
remaining centroids are located in the center of the clusters,
the computation stops (Xu 2004).
Conclusions
Because of falling prices for equipment and oligonucleo-
tides, DNA microarrays are on their way to become
common tools in the microbiological laboratory. They
allow a dynamic view on the physiology of the living cell
and have been compared with a kind of “microscope”
(Brown and Botstein 1999; Ferea and Brown 1999). An
inherent limitation of microarrays is that the resulting
transcriptome does not account for posttranslational events.
However, in most cases, there is a high correlation between
transcriptome and proteome (Akutsu et al. 2000; Hecker
Appl Microbiol Biotechnol (2006) 73:255–273
and Engelmann 2000). The transcriptome data are usually
more comprehensive because of the limited number of
proteins that can be resolved in two-dimensional gels.
Moreover, the relatively detailed knowledge on the genetics
and biochemistry of prokaryotic organisms allows direct
interpretation of the transcriptome data in active pathways.
Many studies have shown that enzyme levels correlate with
their respective gene expression profiles (Arfin et al. 2000;
Smulski et al. 2001; Tao et al. 2001). Future investigations
will show how far this will extend to metabolic flux data
(Kromer et al. 2004; Oh and Liao 2000). The integration of
transcription analysis with comparative genome sequenc-
ing, proteomics, metabolic flux analysis, and computer
modeling of the cell physiology will be an important data
source for system biology, which represents a new
approach for a global quantitative picture of cell physiology
(Galitski 2004). The system biology approach requires a
fundamental framework involving several distinct steps: (1)
definition of all components of a system; (2) systematic
perturbation and monitoring of the components either
genetically or by modification of the environment; (3)
reconcile the experimentally observed responses with those
predicted by a quantitative model; and (4) design and
perform new perturbations to distinguish between multiple
or competing model hypothesis (Ideker et al. 2001).
Transcription analysis is the most important method to
monitor the mRNA abundance of each gene. It will be
complemented by proteomics and metabolomics to provide
the experimental data for model verification as required in
steps 2 and 4 of the outlined strategy (Boyce et al. 2004).
Because system biology will form the foundation of
metabolic engineering, transcription analysis will also be
important in this area (Burja et al. 2003; Vemuri and
Aristidou 2005).
Microarrays can be used in microbiology for a multitude
of differing applications, from the study of gene regulation
and bacterial response to environmental changes, genome
organization, and evolutionary questions up to taxonomic
and environmental studies. The knowledge of the main
aspects of this technology helps to understand these specific
applications.
Vital for further advances of microarray technology in
microbiology will be the recognition of the importance of
the physiological experiments ahead of the transcription
analysis, the standardization of protocols and controls for
transcription analysis (Benes and Muckenthaler 2003),
more integration of the data analysis with biochemical and
genetic knowledge, and flexible and intuitive databases for
mining the vast amounts of data (Mlecnik et al. 2005).
Acknowledgments The author wants to thank Profs. G. Gottschalk,
W. Liebl, and B. Bowien for their support. He is also grateful to Drs. T.
Mascher, P. Ehrenreich, and B. Veith for critically reading the
manuscript. The microarray core facility in the institute of microbiology
Appl Microbiol Biotechnol (2006) 73:255–273
and genetics is part of the competence network Göttingen “Genome
Research on Bacteria” funded by the German Federal Ministry of
Education and Research (BMBF).
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