Biomedical Engineering Reference
In-Depth Information
depends on the extent to which the sequences in the mix complement the DNA affixed to the slide. A
perfect complement, in which a nucleotide sequence in a strand of cDNA exactly complements a DNA
sequence affixed to the slide, will attach more strongly (hybridize) to the DNA sequence than will a
strand of cDNA in which alignment isn't perfect. The strength of adherence, as well as the success in
competing for a spot on the slide, is directly proportional to the degree to which the cDNA and DNA
sequences complement each other.
The populated microarray is then excited by a laser and the resultant fluorescence at each spot in the
microarray is measured. If neither the experimental nor the reference samples hybridize with the
genes at a given spot on the slide—indicating that there are no sequences in either the probe or the
reference that are complementary to the DNA on the slide—the spot won't fluoresce. However, if
hybridization is predominantly with the probe, the spot will be red. Conversely, if hybridization is
primarily between the reference and the DNA affixed to the slide, the spot will fluoresce green. If
cDNA from the probe and reference samples hybridize equally at a given spot—indicating that they
share the same number of complementary nucleotides in the appropriate sequence—the spot will be
yellow. Similarly, various ratios of probe-to-reference hybridization with the slide-mounted DNA
result in colors somewhere in the spectrum between red and green. An analysis of the location,
extent, and exact proportions of red-to-green fluorescence provides a semi-quantitative measure of
gene expression in the tissue sample. That is, even though the fluorescence is digitized and read by
computer, the relative value of the ratios is more exactly determined than is the absolute
fluorescence value, in part because of the variability in the quantity and quality of DNA that is affixed
to the slide during microarray preparation.
An obvious point for the application of statistical methods is at the final stage of the experiment,
where tens of thousands of data points, each indicating relative gene activity, may need to be
analyzed. However, the random variability associated with every stage of the process has to be
considered before the final data can be analyzed in a meaningful way.
A quick check for data validity is to create a scatter plot of fluorescence data from two identically
treated microarrays. As shown in
Figure 6-4
, the ideal condition is when gene expressions as
measured by the microarrays are identical, as indicated by data on the 45-degree ID line, as in (A).
If the amplitude of gene expression on one microarray is greater than the other, data fall off the ID
line, as in (B) and (C). The scatter plot also provides a measure of gene expression amplitude, in that
the greater the distance from the origin, the greater the expression amplitude. For example, the gene
plotted at position (C) has a greater expression amplitude than the gene at position (A).
Figure 6-4. Microarray Results Analysis. Scatter plot illustrating inter-
microarray variability in two identically treated microarrays, Microarray 1
and Microarray 2. Ideally, all data points fall on the ID line, as illustrated by
data point (A).
