Specifications of Xeotron's Human Cancer Xeochips - Invitrogen

Specifications of Xeotron’s
XeoChip TM Gene Expression Bioarray

Xeochip Microarrays
Human Cancer Xeochips were
synthesized in situ using Xeotron’s
patented PGR chemistry on
microfluidic chips. A total of 3728
unique cancer genes are represented on
the Human Cancer Xeochip by single
45 mer oligonucleotide probes. These
were derived from the National Cancer
Institute’s database, CGAP, and
designed based on algorithms to
optimize Tm, CG content and
sequence complexity. In addition, for
these experiments, a Human
Minicancer Xeochip with 252 probes
repeated 15 times for a total of 3780
was synthesized. An additional 384
probes are present as redundancies
designed for housekeeping,
hybridization, and synthesis controls.
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RNA Labeling
Total RNA was purchased from
Clonetech and reprecipitated in ethanol
prior to use. Each experiment was
performed with 100 µg of RNA, 50 µg
labeled with Cy3 dye and 50 µg with
Cy5 dye using Xeotron’s cDNA
labeling kit. Briefly, 50 µg of total
RNA was incubated with oligo dT
primers, dNTP’s reverse transcriptase
and the appropriate Cy dye in buffer at
42° C for 90 min. The RNA was then
removed by hydrolysis with NaOH at
an increased temperature of 65° C for
10 minutes. The resulting labeled
cDNA was purified by elution from
QIAGEN QiaQuick columns in 50 ul
of 0.1X TE. Probe quality was
assessed by measuring the O.D. ratios.
Typically, cDNA with labeling ratios
between 4 and 20 were obtained and
used in the experiments. This
translated into approximately 200 to
400 ng. of labeled cDNA. Xeotron’s
labeling kit is shown in Figure 1.
Figure 1. Xeotron’s cDNA
Labeling Kit

Microarray Hybridization
Prior to hybridization the samples were
pooled and evaporated in a Speed Vac.
BSA (1mg/ml), formamide, and 20
XSSPE were added to make the final
concentration 6X SSPE and 25%
formamide in 100 µl . Control spikes
were added as indicated.
Hybridizations were performed for 18
hours at 32 °C by recirculating the
sample through the microarray using
Xeotron’s Manual Microfluidic
Station. Figure 2A.
Microarray Washing
Washing of the microarrays was
performed by removal of the sample,
followed by circulating 500 µl of 6X
SSPE containing 25% formamide, 500
ul of 6X SSPE and finally 500 µl of
1 X SSPE, sequentially. To stabilize
the helices 250 µl of 6X SSPE was
pumped through the chip for imaging.
The bioarrays were imaged on an
Axon 4000 B scanner at 100% power,
120-micron focus, and PMT adjusted
to the appropriate setting.
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Analysis
Analysis of the arrays was performed
using ArrayPro software (Cybermedia)
and BiochipTools, a Matlab program,
developed in-house. Array Pro was
used to produce the rectangular grid
necessary for feature extraction. Once
extracted, the values were loaded into
the Matlab program for analysis.
Figure 2B. Xeotron’s
BiochipTools for high
throughput data processing and
chip performance evaluation.
Figure 2A. Xeotron’s Manual
Microfluidic Station

These experiments were designed to
determine Xeochip performance when
hybridized under optimal conditions.
The specifications examined included
sensitivity, signal noise, specificity,
and spot variation. Bioarrays were
also examined for probe performance
and accuracy.
Sensitivity,
Linear Dynamic Range, and
These experiments demonstrate the
Xeochip is linear over 4 logs.
To determine the lowest level of
detection (LLD) of the Xeochip, 1% of
the background intensity (excluding
features which contained no
oligonucleotide probe) was subtracted
from the signal intensities. The
concentration of target that could be
detected after this calculation was
Positional Effects 16 fM or approximately 0.96 per 10 6
To measure the sensitivity and linear
dynamic range of the Xeochip, a 45
mer oligonucleotide labeled with Cy3
was used as the target. The
complementary probe was synthesized
in 20 positions throughout the
Xeochip. Figure 3 demonstrates that
signal was detected at values as low as
0.01 pM and the signal was linear to
1000 pM with a regression coefficient
of 0.97.
LogSignal
100000000
10000000
1000000
100000
10000
1000
100
10
(1 copy/cell).
Sensitivity and Linear Dynamic Range
This data further demonstrates that the
reproducibility of hybridization on the
Xeochip is not a function of position.
The c.v. for each group of data was
less than 12% with an average value of
8.8%.
R 2 = 0.9747
1
0.01 0.1 1 10 100 1000 10000
Target Concentration (pM)
Figure 3. Line graph showing the linear dynamic range
of the Xeochip using an oligonucleotide probe labeled
with Cy3 dye.
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System Noise
To evaluate system noise in our
bioarrays, self versus self
hybridizations were performed on the
Human Cancer XeoChip. Significant
deviation from a log ratio of 0 will
lead to false positives. Background
intensities of control features
containing no probe are extremely low
in these arrays; therefore, in order to
increase stringency, 3% of the lowest
signal intensities plus the standard
deviation was subtracted from each
intensity value prior to calculation of
the results. Figure 4 is a representative
histogram showing the log ratios.
Greater than 96% of the population fall
between -0.176 and 0.176 which is
equivalent to a 1.5-fold change in
either direction. Data for this figure
was derived from the log ratio plot
shown in Figure 3.
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Figure 4. Histogram of log ratios for self
versus self experiments. Data shown is
a representative experiment in which
universal RNA was used as the target.
Data is from the XeoEX Human Cancer
bioarray containing 3899 probes shown
in Figure 5.
Figure 5. Representative
log ratio plot of self
versus self experiments
performed with universal
RNA on the Human
Cancer Xeochip. Blue
data points are
statistically similar to
each other and therefore
have a log ratio of 0.
Red data points are
statistically different. All
statistical data is
collected at a
p value < 0.01.

Specificity of Hybridization
Xeotron uses the GAP gene as a
housekeeping control and to monitor
hybridization specificity for human
samples. On each Xeochip, eight
redundant perfect match GAP probes
and GAP probes with three adjacent
mismatches are included. Figure 6
shows the data from 14 independent
experiments.
Even at temperatures as low as 24 °C,
specificity is still extremely precise.
This data indicates that through the use
of Xeotron’s Xeochips and
Microfluidic systems, sequences must
have at least 93% homology to the
probes on the Xeochips before signal
will be detected.
Figure 6. Demonstration of specificity of Xeochip. Plotted are intensity
results from 14 individual experiments over a range of hybridization
temperatures from 24 °C to 42 °C. The top graph is the perfect match probe;
while the bottom is the mismatched probe. Xeochips are now routinely
hybridized at 32 °C in the presence of formamide.
Xeochip Probe Performance
To validate Xeochip probe design three
experiments were performed using
universal RNA labeled with Cy3 and
Cy5. Background was calculated by
first excluding all blanks features. The
lowest 2% of the signal intensities were
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then averaged and the standard deviation
derived. These values were summed and
then subtracted from each individual
intensity. In each case, over 3200
probes showed intensity levels over
background, accounting for greater than
85% of the total number found on the
Human Cancer Xeochip.

Accuracy of Log Ratio
To assess the accuracy of the log ratio,
and ultimately the quality of the data
derived from the Xeochip, a series of
hybridizations were performed with a
synthetic 90 mer transcript prepared in
an in vitro reaction.
The transcript was labeled with either
Cy3 or Cy5 and mixed at the ratios
indicated in the table below. Figure 7
shows the expected and observed log
ratios of the spikes.
Transcript Target Cy3 Target Cy5 Target Spike-in ratio (Cy3:Cy5)
PUC5 5 ng 5 ng 1:1
PUC6 5 ng 20 ng 1:4
PUC25 5 ng 80 ng 1:16
PUC27 5 ng 640 ng 1:128
Figure 7. Titration of Cy3 labeled or Cy5 labeled transcript to measure the
accuracy of the log ratio.
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Xeochip Performance
Spot uniformity is an indication of the
quality of an array. High uniformity is
obtained when the pixel strength is
equivalent throughout the spot. Since
the probes on the Xeochip are
synthesized in situ, Xeochip spots
typically have low c.v. values. Figure 8
demonstrates that spots on any single
XeoChip have, on average, less than
10% c.v. Data averaged from three
Human Cancer XeoChips yielded c.v.
values of 0.045 for the Cy3 channel and
0.065 for the Cy5 channel.
In addition, because the spot
uniformity is so consistent with
Xeochips, they are much easier to grid.
Xeotron provides a grid file that can be
quickly snapped into place. Figure 9 is
an image of a typical chip gridded and
ready for data extraction.
Figure 8. Representative histogram of spot c.v. for (right panel) the Cy3
channel and (left panel) the Cy5 channel. A total of more than 280 genes
were used in these measurements from a Human Cancer Xeochip
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Figure 9. Close-up of a
Xeochip with a grid. Because
of the uniformity of the
Xeochip, A Xeotron supplied
grid is simply overlaid to a tiff
image and pulled into place.

Reproducibility
As a measure of reproducibility, spot-tospot
c.v.s were compared. After
background subtraction, 259 features
were compared on two separate Human
Cancer XeoChips for both the Cy3 and
the Cy5 channel (Figure 10). The
average c.v. for the Cy3 channel was
10% while that for the Cy5 channel was
12%.
Figure 10. Representative histogram showing spot-to spot c.v.
(Right panel) The Cy3 channel had an average c.v. of 10% while
(left panel) the Cy5 channel had an average c.v. of 12%.
Figure 11. Hierarchical clustering analyses for 25 experiments, in
which 12 experiments used universal cDNA for both Cy3 and Cy5 and
13 experiments used skeletal muscle versus brain cDNA. Consistency
of ratio measurements is observed.
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Detection of Differential Expression
To investigate the accuracy of detecting
differential expression, a series of
experiments were performed using
labeled cDNA prepared from either total
brain or skeletal muscle RNA. Five
Minicancer Xeochips were hybridized to
a 1:1 mixture of the labeled cDNA.
Figure 12 is a representative image from
an experiment in which the muscle RNA
was labeled with Cy3 and the brain with
Cy5. To further demonstrate
reproducibility, the color reversal
experiment was performed and the
results are also shown in Figure 13. In
this case the brain cDNA was labeled
with Cy3 while the skeletal muscle was
labeled with Cy5.
Figure 12. (Left panel) Representative log plot of the differential
expression of genes in skeletal muscle RNA (Cy3) vs. brain RNA (Cy5).
(Right panel) Representative log plot of the differential expression of
genes in brain RNA (Cy3) vs. skeletal muscle RNA (Cy5). The red data
points are genes significantly differentially expressed (p

Figure 13. Color reversal experiment demonstrating
reproducibility of Human Cancer Xeochip. Data was derived
from plots above. (Left panel) Log ratio of the skeletal muscle
(Cy3) to brain (Cy5) vs the brain (Cy3) to skeletal muscle (Cy3).
This data has a regression correlation coefficient of 0.89.
(Right panel) Data from separate Xeochips using the labels as
before were compared in silico. In this case the regression
coefficient was 0.96. This demonstrates the reproducibility of
the Xeochip.
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