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LabChip assay (Agilent Technologies, Palo Alto, CA, USA). Utilizing the
GeneChip Whole Transcript Sense Target Labeling Assay kit (Affymetrix,
Santa Clara, CA, USA), 100-300 ng of total RNA undergoes reverse tran-
scription with random hexamers tagged with T7 sequence. The double-
stranded cDNA that is generated is then amplified by T7 RNA polymerase
to produce cRNA. Second-cycle first-strand cDNA synthesis then takes
place, incorporating dUTP, which is later used as sites where fragmenta-
tion occurs by utilizing a uracil DNA glycosylase and apurinic/apyrimidinic
endonuclease 1 enzyme mix. The fragmented cDNA is then labeled by ter-
minal transferase, attaching a biotin molecule using Affymetrix propri-
etary DNA Labeling Reagent. Approximately 2.0
g of fragmented and
biotin-labeled cDNA is then hybridized onto a Mouse Gene ST 1.0 Array
(Affymetrix, Santa Clara, CA, USA) for 16 hours at 45
µ
C. Posthybridization
staining and washing are performed according to the manufacturer's pro-
tocols using the Fluidics Station 450 instrument (Affymetrix). Finally, the
arrays are scanned with a GeneChip Scanner 3000. Images are acquired
and CEL files generated, which are then used for data analysis. Figure 1.3
shows an example of the fluorescence intensities on the chip recorded by
an imaging device.
°
1.2. Experimental Design
Based on our experience, the most critical factor in the experimental design
of a DNA microarray experiment is to have a correct control for compari-
son, which means that we need to appropriately choose a cell or tissue
source for isolating control RNA. Choosing a correct control can be
extremely challenging, and sometimes it may not be possible to find a
“perfect” control. For example, DNA microarray technology has been
widely used in comparing gene expression profiling between tumor cells
or tissues and corresponding normal cells or tissues in humans. Typically,
total RNA is isolated from tumor cells/tissues that are heterogeneous in ori-
gin, and control RNA isolated from cells/tissues adjacent to the tumor
cells/tissues or corresponding normal cells/tissues is also heterogeneous in
most cases; cellular compositions of tumor and normal tissues are not the
same or sometimes not even closely similar for appropriate comparisons.
Although this cellular difference between tumor and normal tissues is a
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