Biomedical Engineering Reference
In-Depth Information
Data Analysis
Once a fluorescence signal is detected, it has to be quantitized or digitized before it can be
manipulated statistically. The digitization or A-to-D conversion is performed at a fixed sampling
frequency, with a converter rated at a certain dynamic range, as measured in bit depth (see
Figure 6-
14
). For example, a 16-bit A-to-D converter can process a signal into one of (2
16
) or 65,636 levels, a
dynamic range of over 4 orders of magnitude—which is generally considered the minimum for gene
expression applications. The output of the digitizer, typically a 16-bit TIFF (.tif) file, is fed to the
workstation for analysis and visualization. One reason that the TIFF format is used over the more
common and space-efficient JPEG (.jpg) format, is that JPEG format uses lossy compression. If data
from the image digitizer are discarded in the compression process, the result is a compressed
dynamic range of the overall system.
Figure 6-14. Analog-to-Digital Conversion. The dynamic range of the
microarray experiment is limited by the resolution or bit depth of the A-to-
D conversion process, as illustrated by the magnified view of the digital
signal.
Analysis of the fluorescence data includes a check for microarray-to-microarray variability using a
scatter plot, as illustrated earlier in
Figure 6-4
. However, assuring microarray-to-microarray
agreement in gene expression levels first assumes that the fluorescence associated with each spot
can be adequately quantified. The most common methods of accomplishing this is to rely on simple
descriptive statistics, such as mean, mode, and median.
The mean is the average pixel density over a spot, corresponding to the average fluorescence
intensity (see
Figure 6-15
). The advantage of using the mean intensity level is that it decreases error
due to variance in DNA deposition during microarray preparation. The mode is the most likely
intensity value, represented by the highest peak in the fluorescence plot. The mode is resistant to
