Biology Reference
In-Depth Information
the location of the spots is facilitated by the fact that the spots are
arranged in a regular array, using either a rectangular grid or a tighter
''orange packing'' layout. Image-processing software is used to define
the location of the spots and to quantify the amount of fluorescence for
each spot. The quantification involves measuring the intensity of the
individual pixels that make up the image of the spot and a separate
measurement of the pixels that make up the background. The
background fluorescence is subtracted from the spot fluorescence, and
the amount of expression is recorded. When the goal is to estimate
the difference between normal and cancer cells, as in our example, the
ratios of red fluorescence (cancer cells) divided by green fluorescence
(normal cells) may be recorded as well. If we were to assign values to the
spots shown in Figure 12-7(A), we might obtain a result such as that
shown in Table 12-1. Spots with no fluorescence are omitted.
Unfortunately, using a ratio means that a gene that has a twofold
increase in expression in the cancer cells, such as gene D, will give
a ratio of 2, but a gene that has a twofold decrease in expression in the
cancer cells, such as gene G, will give a ratio of 0.5. In fact, all of the
decreased expression will be squeezed between 0 and 1, whereas the
increases in expression, such as the 10-fold increase in genes A and H,
can have extremely high numbers. One solution to this problem is to
transform the ratios to logarithms. This is usually done using base 2, as
is shown in Table 12-2.
Fluorescence
*
Spot Position
Ratio
Row
Column
Gene ''Name''
Red
Green
Red/Green
1
2
A
1,000
100
10
1
5
B
120
1,200
0.1
2
1
C
100
1,000
0.1
2
3
D
1,500
750
2
2
4
E
150
1,500
0.1
2
5
F
1,800
1,200
1.5
3
2
G
600
1,200
0.5
3
3
H
800
80
10
4
1
I
1,500
1,500
1
4
3
J
50
500
0.1
4
5
K
1,800
360
5
5
1
L
900
90
10
5
2
M
180
900
0.2
5
4
N
1,000
1,500
0.67
TABLE 12-1.
Synthetic data for Figure 12-7(A).
*
Fluorescence is rarely reported in absolute units. Since the conditions vary widely among experiments because of
equipment differences, the results are given on an intensity scale relative to each experiment.
