323
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3. IMAGE ACQUISITION
The fundamental hybridization reaction,
between two complementary chains, is the basis
to construct gene expression signals.
The microarray image acquisition for the
complete cellular expression profiles studies, is
particularly dependent of the experimental
laboratory protocols followed. The
experimentation information is necessary to
understand completely its repercussions over
final acquisition and final pattern processing
results.
Material, which is deposited on membranes, is
not usually the complete gene sequence but a
part of gene sequence like cDNAs from clone
libraries, like ESTs (at random chosen from
cDNA libraries) (Chen et al, 1998; Bernard et
al, 1996; Schena et al, 1995, 1996) or even like
short oligonucleotides, directly synthesized on
membranes according to sequences which are
determined on the basis of the organism’s
genome (Wodicka et al., 1997; Lockhart et al.,
1996).
Hybridization terms (generally used in this sort
of experimentation) are now introduced. Target
is the cDNA or oligonucleotide molecule which
is deposited on membrane, putative gene is the
reference gene to which cDNA or EST refer,
probe is the mRNA solution hybridizing to the
target. mRNA is extracted, by reverse
transcription, from one (or two) studied cellular
population (populations); during reverse
transcription reaction, mRNA is labeled with
color-forming substances like biotin (blue color)
and digoxigenin (red color) (Chen et al., 1998) or
with fluorescent substances (Schena et al., 1995,
1996; Wodicka et al., 1997; Bernard et al., 1996;
Lockhart et al., 1996). To obtain correct dual
color images, two mRNA amounts, which are
extracted from identical amounts of two cell
populations, must be mixed. On the contrary,
differential gene expressions could be read
erroneously and could be confused with the
simple difference of amount between the two
probes.
(Chen et al., 1998) used a PC-controlled XYZ
translation system fitted with steel pins. This
arraying machine spotted cDNA fragments on a
nylon membrane and the deposition volume was
governed by the diameter of the tips. This
system is able to place 100-pm-diameter cDNA
spots with spacing of 150 pm (with a cDNA
quantity of 10 ng). Therefore it is estimated that
about 85,000 spots can be placed on a nylon
membrane measuring (35x55) mm. In
consequence of hybridization reaction, the
membrane is treated with suitable reagents and
washed; color-forming reaction can develop for
those probe molecules, which hybridized to
target molecules.
(Wodicka et al, 1997) synthesized, for the whole
genome of Saccharomyces cerevisiae (6200 genes),
oligonucleotides, of known sequence, directly,
on membrane. The authors produced four
arrays, measuring (1.28x1.28) cm, containing
more than 65,000 features of (50x50) pm. In this
case fluorescence labeling occurs after the
hybridization reaction and after stripping of not-
hybridized material.
In the absence of any sort of error, the signal
intensity of the generic spot is proportional to
the expression of the target in the cellular
population.
Therefore the signal is ready to be digitized by
means of tools available in any research
laboratory: flatbed scanners (the least expensive
scanners with a resolution of 600 dpi), drum
scanners (with a resolution of 3000 dpi) (Chen et
al., 1998), color video cameras (Chen et al,
1998), digital cameras attached to a
stereomicroscope (to digitize spots with
diameter of 100 pm or less thanks to the high
resolution of the device) (Chen et al, 1998),
scanning confocal fluorescence microscopes with
a resolution of the order of 10 pm (Wodicka et
al, 1997; Lockhart et al., 1996) and scanning
laser-inducted fluorescence devices (Schena et al,
1996). Single-color images are then converted
into grey level images. Dual-color images,
produced by fluorescence, are converted into
pseudo-color images by means of suitable
software.
Specificity and uniqueness of selected targets,
compared with organism’s genome, is very
important, even though in the case of cDNA
targets this requirement is not always warranted.
Stripping (with proper substances) of the labeled
material and subsequent washing allow the
reutilization of the nylon membrane. In the
studies of (Chen et al, 1998) the nylon
membrane can be used four times without
degradation of the signal-noise ratio. The two
gene expression image construction schemes and
an example of image are shown in figures 2, 3
and 4.
