Biomedical Engineering Reference
In-Depth Information
The first source of variability to consider—a shift in relative location of a spot in the microarray—is
problematic for several reasons. First is that when a microarray is scanned by a laser, a displaced
spot won't read as strongly as it would otherwise because part of the spot may be outside of the field
of the exciting laser beam. This type of variability can be addressed somewhat if starring is used to
read the microarray because image-recognition technology can be used to identify spot location on
the captured image. Image-recognition software can search the immediate vicinity where a spot is
expected and appropriately adjust the location of the image pixels that are read.
Variations in spot density due to uneven adherence of cDNA to the slide during the spotting process
may result in erroneous output signal interpretation, depending on the statistical method used to
analyze spot intensity. Variations in spot shape and deviations from expected spot location result in
errors in intensity reading of spot fluorescence. Starring and scanning are both susceptible to
variations in spot shape. In starring, a mask is used to limit the extent of the area read on the
captured image, even though the excitatory laser beam covers many spots at once. The opposite is
true of scanning, in that the image capture device is receptive to fluorescence signals from anywhere
on the microarray. However, only a small area of the array is excited at a time. As a result, a
misshapen spot may not contribute fully to its expected fluorescence intensity.
Contamination of the microarray, whether from dust or extraneous organic material in the slide
coating, is another source of variability that is difficult to counteract. Contamination can interfere
with automated spot-locating technologies used with starring and partially obscure spots in the
microarray so that they can't be properly scanned. Contamination can also give false positive
indications of the level of gene expression when it is highly fluorescent and falls on spots that would
otherwise not fluoresce.
At a higher level is variability due to the overall system operation and processes. Sources of process
variability include photobleaching, in which the exciting laser or other light bleaches the fluorescing
dye, rendering the scanned spots useless for subsequent analysis. Photobleaching, which is a
function of laser intensity and the time the laser dwells on each spot, is problematic when the
microarray reading process is interrupted mid-way through a reading cycle and re-started. The
previously excited area on the microarray may be faded relative to the unread area in the
microarray. This may be problematic when gene activity corresponding to dual or multiple excitations
is compared with areas of single excitation.
In addition to process variation, there is also variability due to the particular equipment used in the
microarray system. Some of this variability or noise may be due to improper design or because of
overwhelming noise level in the environment. For example, data acquisition devices are subject to
