323 
e. This reaction is 
It is evident how it 
DNA chain (or a 
back to the 
;ment). 
le whole genetic 
which means that 
the development 
s located in the cell 
Dtality of the living 
is the cell different 
fen from itself in 
is the expression 
ch genes express 
>utes itself along 
mes. Genes are 
nd the position of 
, on the respective 
contained in the 
C, G, T) along the 
lg gene sequence). 
by introns (not- 
iquencing means 
e nucleotide bases 
ie. 
messenger RNA 
linucleotide chain, 
e two DNA chains 
ig strand”). During 
eliminated and the 
mslates into the 
ence according to 
.ch mRNA codon 
is to a particular 
espondence. 
is determined by 
: produced by gene 
.s) are greatly used 
;le radioactively or 
A chains allowing 
ry DNA sequence 
)rial chemistry for 
id from mRNA 
iption. Pieces of 
d ESTs (Expressed 
;ed for expression 
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.