Each spot on a DNA microarray contains thousands of oligonucleotide
molecules called probes that complement their corresponding mRNA. A
sample is prepared for DNA microarray analysis by extracting the RNA
from the cells. The RNA can be purified by treating it with poly(A) polymerase followed by oligo(dT) chromatography. The purified RNA is then
labeled for detection; in the past,
33
P has been used, although radioactive
labeling has mostly been replaced by the use of fluorescent markers such
as the cyanine dyes Cy3 and Cy5. The labeled RNA “targets” are then
spotted on the DNA microarray probes to which the RNA binds in a
process called “hybridization.” Before measuring fluorescence, the DNA
microarray is washed to remove any excess or unhybridized targets.
Fluorescence can be quantified by confocal laser scanning microscopy or
other fluorescence microscopy techniques. Using internal standards,
fluorescence can be used to calculate the number of targets that hybridize
to the probes, which can then be used to determine the change in gene
expression in a normal state compared to an experimental state. Running a
DNA microarray experiment is relatively quick and easy; the biggest factor
detracting from more widespread use is the lengthy fabrication process.
10.7.2 Array fabrication
There are three primary methods for DNA microarray fabrication: in situ
synthesis, contact printing, and noncontact printing (Dufva et al., 2005). In
situ synthesis creates the oligonucleotides directly on the microarray and is
capable of producing the highest quality microarrays, although these highquality arrays require expensive robotics systems and the use of clean room
techniques. All methods of in situ synthesis use nucleosides that have
already been modified with a protecting group to ensure that the correct
sequences of oligonucleotides are present on completion. In the first
method (Figure 10.24a), nucleosides are modified with the photolabile
reagent 2-nitrophenyl propoxycarbonyl (NPPOC). These protecting groups
are removed using light, which allows the growing oligonucleotides on the
array react with available nucleosides in solution. Synthesis is controlled by
the use of various masks that are placed on top of the microarray, allowing
only specific regions to react. After subjecting a mask to light, the appropriate nucleoside is added, the microarray is washed, and then the process
repeats. The downside to this method is that it requires a different mask for
each step of the synthesis. Another method (Figure 10.24b) involves
similar chemistry, yet instead of masks, a series of mirrors is used to direct
light to the spots to be deprotected. Yields of 77% for 25 base pair oligonucleotides have been achieved using NPPOC as the protecting group.
DNA AND LIPID MICROARRAYS 421
molecules called probes that complement their corresponding mRNA. A
sample is prepared for DNA microarray analysis by extracting the RNA
from the cells. The RNA can be purified by treating it with poly(A) polymerase followed by oligo(dT) chromatography. The purified RNA is then
labeled for detection; in the past,
33
P has been used, although radioactive
labeling has mostly been replaced by the use of fluorescent markers such
as the cyanine dyes Cy3 and Cy5. The labeled RNA “targets” are then
spotted on the DNA microarray probes to which the RNA binds in a
process called “hybridization.” Before measuring fluorescence, the DNA
microarray is washed to remove any excess or unhybridized targets.
Fluorescence can be quantified by confocal laser scanning microscopy or
other fluorescence microscopy techniques. Using internal standards,
fluorescence can be used to calculate the number of targets that hybridize
to the probes, which can then be used to determine the change in gene
expression in a normal state compared to an experimental state. Running a
DNA microarray experiment is relatively quick and easy; the biggest factor
detracting from more widespread use is the lengthy fabrication process.
10.7.2 Array fabrication
There are three primary methods for DNA microarray fabrication: in situ
synthesis, contact printing, and noncontact printing (Dufva et al., 2005). In
situ synthesis creates the oligonucleotides directly on the microarray and is
capable of producing the highest quality microarrays, although these highquality arrays require expensive robotics systems and the use of clean room
techniques. All methods of in situ synthesis use nucleosides that have
already been modified with a protecting group to ensure that the correct
sequences of oligonucleotides are present on completion. In the first
method (Figure 10.24a), nucleosides are modified with the photolabile
reagent 2-nitrophenyl propoxycarbonyl (NPPOC). These protecting groups
are removed using light, which allows the growing oligonucleotides on the
array react with available nucleosides in solution. Synthesis is controlled by
the use of various masks that are placed on top of the microarray, allowing
only specific regions to react. After subjecting a mask to light, the appropriate nucleoside is added, the microarray is washed, and then the process
repeats. The downside to this method is that it requires a different mask for
each step of the synthesis. Another method (Figure 10.24b) involves
similar chemistry, yet instead of masks, a series of mirrors is used to direct
light to the spots to be deprotected. Yields of 77% for 25 base pair oligonucleotides have been achieved using NPPOC as the protecting group.
DNA AND LIPID MICROARRAYS 421