Note: Descriptions are shown in the official language in which they were submitted.
CA 02485055 2005-06-23
IN SITU DILUTION OF EXTERNAL CONTROLS FOR USE IN MICROARRAYS
FIELD OF THE INVENTION
The present invention is related to the field of arrays more specifically to
array
systems.
BRIEF DESRIPITON OF THE PRIOR ART
DNA arrays are commonly used to make quantitative or relative measurements of
gene expression. They provide a medium for matching known and unknown DNA
samples based on base-pairing rules and automating the process of identifying
the
unknowns. In general, arrays are described as macroarrays or microarrays, the
difference being the size of the sample spots. Macroarrays contain sample spot
sizes of
about 300 microns or larger and can be easily imaged by existing gel and blot
scanners.
The sample spot sizes in microarrays are typically less than 200 microns in
diameter and
these arrays usually contains thousands of spots.
The microarrays contain nucleotide sequences corresponding to known genes or
expressed sequence tags. A single microarray can contain thousands of genes,
which
may represent a significant subset of the genes, or even the entire genome, of
an
organism. A comparison of cells or tissues from experimental and control
preparations
provides data on differences in expression levels between the two conditions.
For this
purpose, mRNA is extracted from a sample, converted to complementary DNA
(cDNA)
and tagged with a fluorescent label. In a typical microarray experiment, cDNA
from one
sample (sample A) is labeled with a first dye that fluoresces in the red and
cDNA from
another sample (sample B) is labeled with a different dye that fluoresces in
the green.
The fluorescent red and green cDNA samples are then applied to a microarray
that
contains DNA fragments (oligonucleotides) corresponding to thousands of genes.
If a
DNA sequence probe is present on the microarray and its target complement is
present
in one or both samples, the sequences bind, and a fluorescent signal can be
detected at
the specific spot on the array. The signals are generally picked up using a
"scanner"
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which creates a digital image of the array. The red to green fluorescence
ratio in each
spot reflects the relative expression of a given gene in the samples A and B.
Current microarray analyses rely on normalization and quality control methods
that
often assume evenly distributed changes, and/or absence of global shifts in
gene
expression across the array surface. Spotted microarray features such as
housekeeping
genes, sample pools, genomic DNA, or all genes on a microarray are typically
used for
normalization. Normalization based on these features is not always
appropriate,
especially for smaller focussed arrays (versus whole genome microarrays) where
unbalanced changes are likely to occur, and will have significant effects on
the relative
hybridization signal intensities between biological samples. As a result,
normalization
based on such features will give rise to inaccurate interpretations of gene
expression
data.
According to W02004/064482, normalization and quality assessment of
microarray data, where unbalanced gene expression is anticipated, can be
accomplished by the addition of several different external, non-species
nucleic acid
targets of different concentrations into the RNA sample of interest prior to
labelling and
hybridization. Different concentrations of external control targets are chosen
to mimic a
broad range of expression profiles. Probes complimentary to the external
targets are
printed at equivalent concentrations on the microarray. Variation between
external
control target concentrations in the sample results in different fluorescence
intensities
detected for each external control probe. Since detection of the different
external
controls will be equivalent between RNA samples, and are not affected by
unbalanced or
global shifts in gene expression within the RNA sample of interest, they can
be used for
accurate normalization and interpretation of gene expression data from
focussed
microarrays.
The drawback to using an external control, where varying amounts of different
targets are added to the RNA sample of interest, is that it requires accurate
measurement of extremely small quantities of those several RNA targets at low
concentrations. The technical error associated with measurements at the low
range
required for microarray analysis results in unacceptable variation between
samples that
will have a significant influence on normalization and interpretation of gene
expression
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data. In addition, the optimization and preparation of multiple external
control targets and
probes is time-consuming and costly.
Accordingly, there is a need for a microarray system that allows for more
accuracy in the normalization of the data.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method that satisfies the
above-
mentioned need.
More particularly, the present invention provides a microarray and a
hybridizing
reagent that allow for more accuracy in the normalization of data.
The present invention teaches the addition of at least one external non-
species
nucleic acid or protein target, which can be accurately measured, to each
biological
sample prior to labelling and hybridization and the concurrent use of a
matched external
probe (either DNA, RNA or antibody) printed on the microarray in a series of
different
concentrations. The dilution of a control probe in situ (i.e. printed on the
hybridization
surface in different concentrations) effectively mimics a broad range of
expression
profiles. This approach allows an experimenter to apply at least one external
control
target spiked into RNA or protein samples reducing the preparation time an
cost
normally associated with the development and use of external controls.
Variation in the amount of printed probe will result in variation of the
amount of
hybridized external control target resulting in the detection of a broad range
of
hybridization signal intensities. Since the amount of external control target
added to each
biological sample is equivalent, and unaffected by unbalanced or global shifts
in
expression within the sample, detection of the external control will be
equivalent across
all samples. These external control features can then be utilized for
normalization,
quality control, validation or other applications.
The addition of a constant and an accurately measurable quantity of external
control target to each biological sample prior to labelling and hybridization,
combined
with the printing of several different concentrations of the external control
probe covering
the dynamic range of signal detection, represents a novel approach to
microarray
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normalization, quality control, validation and other applications that is
unaffected by
unbalanced or global shifts in gene expression.
The present invention presents the advantage of not relying on pipetting
different
concentrations and volumes of external DNA/RNA into reaction mixtures and is
therefore
much more accurate.
The present invention also has the advantage of providing means of carrying
out
in situ dilution in microarrays that are less expensive to operate and are not
time
consuming. In addition, the present invention presents the advantage of
providing means
of carrying out in situ dilution in microarrays used in toxicological studies
or in comparing
two states of a cell or in a study requiring the use of arrays or microarrays.
The present invention thus provides for an array system comprising:
a solid support having at least one grid;
at least one external control probe attached to at least one grid of the solid
support in a series of concentrations, the concentrations are randomized in
each grid;
a hybridizing mixture containing at least one external control target
complementary to the external control probe; and
a plurality of sample probes attached to the solid support at discrete
locations.
The present invention also provides a process of normalizing an array system,
comprising the steps of:
a) providing a solid support comprising at least one grid;
b) attaching at least one external control probe to said solid support in a
series of
concentrations, each of said series of concentration of said at least one
external control
probe being attached to a grid of said solid support at a different discrete
location from
another concentration of said series, said series of concentrations being
randomized in
said grid;
c) attaching at least one sample probe on at least one grid to said solid
support;
d) making several grids by repeating steps a) and b);
e) hybridizing the hybridizing mixture to the microarray;
f) measuring level of hybridization between the at least one external control
probe
and the at least one external control target and between the plurality of
sample probes
and plurality of sample targets; and
g) determining amount of sample target present in the hybridizing mixture.
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The present invention also provides a kit comprising:
a solid support having at least one grid;
at least one external control probe attached to at least one grid of said
solid
support in a series of concentrations randomized in each grid;
a hybridizing mixture containing at least one external control target
complementary the external control probe; and
a plurality of sample probes attached to the solid support at discrete
locations.
Other objects and advantages of the present invention will be apparent upon
reading the following non-restrictive detailed description, made with
reference to the
accompanying drawings
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a grid layout according to the invention.
Figures 2A, B and C show other grid layouts according to the invention.
Figure 3 shows a diagrammatic representation of microarray printing with ~rTek
ChipWritePro ~ (BioRad) using 12 pins.
Figure 4 shows the ToxArray~ genes sorted by ontology.
Figure 5 is images of identical hybridizations of oligo test set on two slide
types
showing (arrows) the increase in detectable test oligo spots on the
PowerMatrix~ slide
(a) compared to the QMT Epoxy~ slide (b).
Figure 6 shows the effect of oligo spotting buffer on mean spot intensity.
Figure 7 shows a partial microarray image of Hsp84-1 oligo printed in 4
different
printing buffers on the PowerMatrix~ slide.
Figure 8 shows the effect of oligo printing concentration on signal
intensities. A is
the logarithmic plot of mean spot intensities of a subset of test oligos
printed at different
concentrations. B is the image of a subset of RpL5 oligo printing
concentration.
Figure 9 shows a typical microarray image of oligos test set printed at 40 pM,
in
ArraylT~ spotting solution (Telechem International) on the PowerMatrix~ slide
(Full
Moon Biosystems).
Figure 10 shows the outline of an externally controlled microarray experiment.
Figure 11 shows the mean signal intensities of external control dilutions and
test
oligo features.
Figure 12 shows the external control dilution series spot intensities. A shows
the
logarithmic plot of external dilution series spot intensities for 5
independent microarray
experiments (Cy5 and Cy3). B shows a subset of external control features from
a typical
microarray image.
Figure 13 shows signal intensities for a single external control dilution
sorted by
array grid position.
Figure 14 shows signal intensities for a single external control dilution
sorted by
printing pin.
Figure 15 shows a linear plot of external control dilution series.
Figure 16 shows replicate external control spots sorted by grid and
metacolumn.
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LIST OF TABLES
Table 1 is a list of positive control house keeping genes.
Table 2 is a list of the commercial glass slides tested.
Table 3 is a list of the oligonucleotides printing buffers tested.
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DETAILED DESCRIPTION OF THE INVENTION
Definitions:
In order to provide an even clearer and more consistent understanding of the
description, including the scope given herein to such terms, the following
definitions are
provided:
Gene refers to nucleic acid sequences (including both RNA or DNA) that encode
genetic information for the synthesis of a whole RNA, a whole protein, or any
portion of
such whole RNA or whole protein.
Probe refers to a nucleotide sequence often an oligonucleotide that is, or is
intended to be, attached to a solid support in an array that can be used to
hybridize with
and thereby identify the presence of a complementary sequence, or a
complementary
sequence differing from the probe sequence but not to a degree that prevents
hybridization under the hybridization stringency conditions used. As is well
known to
those skilled in the art, for the hybridization characteristics to be similar
across a wide
range of oligonucleotides, it is typically required that the probes on the
array be of the
substantially same length, have a similar percentage of Guanine to Cytosine
content and
lack any extensive runs of poly A, poly G, poly C, or poly T tracts. The goal
of controlling
these parameters is to produce probes that have similar melting and
hybridization
temperatures. Additionally, these probes should, preferably, lack length
complementary
regions and not form hairpin structures. The probe can be DNA, RNA,
oligonucleotides,
cDNA, proteins or antibodies.
Target refers to nucleic acids intended to be hybridized (or bound) to probes
immobilized on microarrays by sequence complementarity. The target can be DNA,
RNA, oligonucleotides, cDNA, proteins or antibodies. As is well-known in the
art, target
nucleic acids may be obtained from a wide variety of organisms (bactera,
plants...),
tissues or cells. Methods and techniques for the extraction, manipulation and
preparation
of nucleic acids for hybridization reactions are well-known in the art (see,
for example, J.
Sambrook et al.,"Molecular Cloning : A Laboratory Manual", 1989, 2nd Ed. ,
Cold Spring
Harbour Laboratory Press: New York, NY ;"PCR Protocols : A Guide to Methods
and
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Applications", 1990, M. A. Innis (Ed. ), Academic Press: New York, NY;
P.Tijssen"Hybridization with Nucleic Acid Probes- Laboratory Techniques in
Biochemistry andMolecular Biology (Parts I and II)", 1993, Elsevier
Science;"PCR
Strategies", 1995, M. A.Munis (Ed. ), Academic Press: New York, NY;and"67
Protocols
in Molecular Biology", 2002,F. M. Ausubel(Ed. ), 5h Ed. , John Wiley & Sons).
Complement when used in reference to a given polynucleotide sequence refers
to a sequence of nucleotides which can form a double-stranded heteroduplex in
which
every nucleotide in the sequence of nucleotides is base-paired by hydrogen
bonding to a
nucleotide opposite it in the heteroduplex with the given polynucleotide
sequence. The
term may refer to a DNA or an RNA sequence that is the complement of another
RNA or
DNA sequence. As used herein, the term "hybridizes" refers to the formation of
a
hydrogen-bonded heteroduplex between two nucleic acid molecules. Generally, a
given
nucleic acid molecule will hybridize with its complement, or with a molecule
that is
sufficiently complementary to the given molecule to permit formation of a
hydrogen-
bonded heteroduplex between the two molecules.
Oligonucleotide Oligonucleotides means nucleic acid, either desoxyribonucleic
acid (DNA), or ribonucleic acid (RNA), in single-stranded or double-stranded
form and
having one nucleotide or more whether, occurring naturally or non-naturally in
a
particular cell, tissue or organism, and any chemical modifications thereof.
Such
modifications include, but are not limited to providing other chemical groups
that
incorporate additional charge, polarizability, hydrogen bonding or
electrostatic interaction
to one or more of nucleic acid bases of the oligonucleotide.
Specifically hybridizing refers to the association between two single-stranded
nucleotide molecules of sufficiently complementary sequence to permit such
hybridization under pre-determined conditions generally used in the art
(sometimes
termed "substantially complementary"). In particular, the term refers to
hybridization of
an oligonucleotide with a substantially complementary sequence contained
within a
single-stranded DNA or RNA molecule of the invention, to the substantial
exclusion of
hybridization of the oligonucleotide with single-stranded nucleic acids of non-
complementary sequence.
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Toxicant refers to any substance potentially toxic. It also refers a chemical
having
the qualities or effects of a poison and to a harmful substance or agent that
may injure
an exposed organism. Toxicants acccording to the pesent invention may be
chemical
toxicants like for instance cyanides, phenols, pesticides, or heavy metals.
Toxicants may
also be physical in nature like for instance asbestos, silicas and they may
also be
radioactive.
Normalization refers to the process of removing superfluous differences in
array
or microarray data by reducing them to a common denominator.
External refers to a molecule that does not hybridize with the molecules of
the
collection or sample under study. In a preferred embodiment, a nucleic acid
sequence is
thus "external" if its complement is not present in the nucleic acids of a
collection. The
source or collection may preferably be a plurality of nucleic acids to be
hybridized to an
array. The external control probe has a sequence that does not hybridize to a
target from
the plurality of targets from an organism under study to be hybridized on an
array or
microarray.
Hybridizing mixture refers to a mixture being or intended to be hybridized to
an
array. Those of ordinary skill in the art will appreciate that the hybridizing
sample may
contain DNA, RNA, or both, but most commonly contains cDNA. The hybridizing
mixture
typically contains nucleic acids whose hybridization with probes on an array
is
detectable. For example, in many embodiments, the hybridizing mixture
comprises or
consists of detectably labelled nucleic acids.
Labelled, Detectably labelled, labelled with a detectable agent: specify that
a
nucleic acid molecule or individual nucleic acid segments from a sample can be
detected
and/or visualized following binding (i. e. hybridization) to probes
immobilized on an array.
The detectable agent is such that it generates a signal which can be measured
and
whose intensity is related to the amount of hybridized nucleic acids. The
detectable
agent is such that it generates a localized signal, thereby allowing spatial
resolution of
the signal from each spot on the array. Methods for labelling nucleic acid
molecules are
well known in the art. Suitable detectable agents include, but are not limited
to: various
CA 02485055 2005-06-23
ligands, radionuclides, fluorescent dyes, chemiluminescent agents,
microparticles,
enzymes, colorimetric labels, magnetic labels, and haptens.
Fluorescent label refers to a molecule, which, in solution and upon excitation
with
light of appropriate wavelength, emits light back. Numerous fluorescent dyes
of a wide
variety of structures and characteristics are suitable for use in the practice
of this
invention. Similarly, methods and materials are known in the art for
fluorescently
labelling nucleic acids (see, for example, R. P.Haugland,"Molecular Probes :
Handbook
of Fluorescent Probes and Research Chemicals 1992-1994", Sti' Ed. , 1994,
Molecular
Probes, Inc.). In choosing a fluorophore, it is generally preferred that the
fluorescent
molecule absorbs light and emits fluorescence with high efficiency (i. e., it
has a high
molar absorption coefficient and a high fluorescence quantum yield,
respectively), and is
photostable (i. e. it does not undergo significant degradation upon light
excitation within
the time necessary to perform the array-based hybridization). Suitable
fluorescent labels
for use in the practice of the methods of the invention include, for example,
Cy-3, Cy-5,
Texas red, FITC, Spectrum Red, Spectrum Green, Alexa-488, phycoerythrin,
rhodamine,
fluorescein, fluorescein isothiocyanine, carbocyanine, merocyanine, styryl
dye, oxonol
dye, BODIPY dye, and equivalents, analogues or derivatives of these molecules.
Array refers to an arrangement on a solid support of multiple nucleic acid
molecules of known or unknown sequences. These nucleic acid molecules are
attached
to discrete "spots" or positions on the support. A discrete spot may contain a
single
nucleic acid molecule or a mixture of different nucleic acid molecules. Spots
on an array
may be arranged on the support surface at different densities. In general,
microarrays
with probe pitch smaller than 500 pm (i. e., density larger than 400 probes
per cm2) are
referred to as high density microarrays, otherwise, they are called low
density
microarrrays. Arrays come as two-dimensional probe matrices (or supports),
which can
be solid or porous, planar or non-planar, unitary or distributed. The term
"microarray"
more specifically refers to an array that is miniaturized so as to require
microscopic
examination for visual evaluation. Arrays used in the invention are preferably
microarrays.
Expression refers to the process by which nucleic acid is transcribed into
mRNA
and translated into peptides, polypeptides, or proteins.
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Nucleic acid refers to either desoxyribonucleic acid (DNA), or ribonucleic
acid
(RNA).
Polynucleotide refers to any DNA, RNA sequence or molecule having one
nucleotide or more, including nucleotide sequences encoding a complete gene.
The
term is intended to encompass all nucleic acids whether occurring naturally or
non-
naturally in a particular cell, tissue or organism. This includes DNA and
fragments
thereof, RNA and fragments thereof, cDNAs and fragments thereof, expressed
sequence tags, artificial sequences including randomized artificial sequences.
Kit may comprise packages, each containing one or more of the various reagents
(typically in concentrated form) required to perform the respective tests. The
kits
according to these embodiments of the invention are contemplated to be useful
for
detecting and/or quantifying DNA, RNA, and/or protein in biological (or other
types of)
samples.
GENERAL OVERVfEW OF THE INVENTION
The present inventors have discovered that it is possible to normalize
microarrays by using external control probes in a series of dilutions across
the
microarray itself. Therefore the present invention is directed towards an
array or a
microarray having a series of dilutions of an external probe attached to it, a
process of
normalization by using such a series of concentrations of an external probe
and also a
kit comprising the array containing the series of concentrations of the
external probe and
a hybridizing buffer containing the external control target.
Other objects and advantages of the present invention will be apparent upon
reading the following non-restrictive description of several preferred
embodiments and
the accompanying examples.
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DESCRIPTION OF PREFERRED EMBODIMENTS
The inventors have found a way to normalize an array or a microarray by using
a
series of concentrations of an external control probe spanning all areas of a
grid in the
array or microarray. Thus the present invention provides for an array system
comprising:
a solid support having at least one grid;
at least one external control probe attached to the grid in a series of
concentrations which is randomized in the grid;
a hybridizing mixture containing at least one external control target
complementary to the external control probe; and
a plurality of sample probes attached to the solid support at discrete
locations.
According to a preferred embodiment of the invention, the array system is a
microarray system.
In a preferred embodiment, the sample probes and the external control probes
are printed on the solid support. In a more preferred embodiment the printing
is done by
a Virtek ChipWriterPro~ (BioRad). According to another preferred embodiment of
the
invention, the Virtek ChipWriterPro~ has 12 pins, and as can be seen in figure
1, each
pin produces a distinct grid with x rows and y columns. The external control
probe is a
molecule that does not hybridize with the molecules of the collection or
sample under
study. This in situ approach of printing of the series of concentrations of
the external
control probe, does not rely on pipetting different concentrations and volumes
of different
RNA/DNA into reaction mixture and is therefore much more accurate.
The array system according to the invention is further characterized by a
hybridizing mixture, which comprises a plurality of sample targets to be
hybridized on the
array. According to a preferred embodiment of the invention, the external
control probe
and the sample probes of the array are chosen from DNA, RNA, oligonucleotides,
cDNA,
proteins and antibodies and more preferably they are oligonucleotides, and
even more
preferably 70mer oligonucleotides. The external control probe may be from a
bacterial,
plant, animal gene or from any other organism. In a preferred embodiment of
the
invention, the external control probe is an oligo probe designed for the
Arabidopsis
thaliana chlorophyll synthase gene and the external control target is RNA
prepared
through in vitro transcription of A. thaliana chlorophyll synthase cDNA.
The concentration of the external control probe is chosen to start at
extremely low
molarity and going up to saturation. These concentrations are chosen to mimic
the range
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of the expression profile of the studied sample and allow the control for
differential
hybridization occurring across the chip. In a preferred embodiment of the
invention, the
series of concentrations of the external control probe varies from 0.0015 to
100 NM. In a
more preferred embodiment of the invention, the series of concentrations of
the external
control probe is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244, 0.0488, 0.0077,
0.195, 0.39,
0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 pM. As can be
appreciated from
figures 2A and 2B, showing preferred embodiments of the invention, the series
of
concentrations is spatially randomized and duplicated on a grid. A grid also
contains at
least one spot with buffer, spots that are left empty and at least one spot
printed with a
housekeeping gene, the house keeping gene probe being preferably RpLS.
As mentioned above, the sample probes are chosen from DNA, RNA,
oligonucleotides, cDNA, proteins and antibodies. These samples probes are
representative of relevant genes under study. In a preferred embodiment, the
relevant
genes under study are toxicologically relevant genes. According to a preferred
embodiment, the sample probes are oligonucleotides. In another preferred
embodiment
of the invention the sample probes oligonucleotides are 70mer
oligonucleotides. In yet
another preferred embodiment of the invention, the 70mer oligonucleotides are
designed
and synthesized according to the following characteristics:
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
- less than 20% cross homology; and/or
- less than 20 contiguous bases in common with top BLAST hit gene.
The invention is further characterized in that the sample probes are attached
to
the solid support in a fixed concentration. Hence, the 70mer oligonucleotides
of the
invention are attached to the solid support in a fixed concentration. In a
preferred
embodiment of the invention, the 70mer oligonucleotides are attached to the
solid
support at a concentration chosen from 60, 40, 30, 15, 7.5 and 3.5 NM and
preferably at
the concentration of 40NM. In a preferred embodiment of the invention, the
70mer
oligonucleotides are representative of toxicologically relevant genes in
regulatory
toxicology and risk assessment studies.
As can be appreciated in figure 1, according to a preferred embodiment of the
invention, the array system is further characterized in that the series of
concentrations is
spatially randomized across a grid and attached in duplicate in each grid of
the array.
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Having at least two copies of different dilutions of the external control
spanning the area
across a chip, allows the control of differential hybridization. As can be
appreciated also
from figure 1,
The array system is further characterized in that the series of concentrations
of
the external control probe varies from 0.0015 to 100 NM. More preferably the
series of
concentrations of the at least one external control probe is 0.0015, 0.0031,
0.0061,
0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5,
25, 50, 60,
80 and 100 NM.
The invention also provides for the process of normalizing an array system,
comprising the steps of:
a) providing a solid support comprising at least one grid;
b) attaching at least one external control probe to the solid support in a
series of
concentrations, each concentration of the series is attached to a grid of the
array at a
different discrete location, the concentrations are also randomized in the
grid;
c) attaching sample probes spatially arranged in the grid ;
d) making several grids on the solid support by repeating steps a) and b);
e) hybridizing the hybridizing mixture to the microarray;
f) measuring level of hybridization between the at least one external control
probe and
the at least one external control target and between the plurality of sample
probes and
plurality of sample targets; and
g) determining amount of sample target present in the hybridizing mixture.
In a preferred embodiment of the invention the series of concentration of the
external control probe of the process varies from 0.0015 to 100 NM and even
more
preferably it is is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244, 0.0488, 0.0077,
0.195, 0.39,
0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 NM.
In another preferred embodiment of the invention the sample probes and the
external control probes are from DNA, RNA, oligonucleotides, cDNA, proteins
and
antibodies, more preferably oligonucleotides. In a preferred embodiment of the
invention
the sample probes of the process are 70mer oligonucleotides, more preferably
designed
and according to the following characteristics:
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
CA 02485055 2005-06-23
- less than 20% cross homology; and/or
- less than 20 contiguous bases in common with top BLAST hit gene.
According to a preferred embodiment of the invention, the process is further
characterized in that it comprises the following step:
h) attaching the at least one external control probe series of concentration
in duplicate in
the grid.
In another preferred embodiment of the invention, the process is further
characterized in that it comprises at least one of the following steps:
i) adding a buffer only to at least one discrete spot on the grid;
j) leaving at least one discrete spot in said grid empty;
k) attaching a random hexamer to one spot on said grid;
I) attaching a random pool of 70mer oligonucleotides to at least one discrete
spot on said
grid; and
m) attaching a probe from a housekeeping gene to at least one discrete spot on
said
grid.
In a preferred embodiment of the invention, the 70mer oligonucleotides are
attached to the solid support at a concentration chosen from 60, 40, 30, 15,
7.5 and 3.5
NM and more preferably at 40 NM.
The composition and the method of preparation of the buffer of step e) is well
known in the art. In a preferred embodiment of the present invention, the
buffer is PBS~,
Quantifoll I~, Quantifoll II~ and ArrayIT~, and more preferably ArrayIT~. In a
preferred
embodiment of the invention, the housekeeping gene is RpLS.
In a preferred embodiment of the invention, and as can be seen from figures
2A,
2B, and 2C, the buffer can be added to one spot, 6 or 7 spots in the grid.
Also in a
preferred embodiment of the invention, no spot or one or 4 spots of the grid
are left
empty. In a preferred embodiment, the house keeping gene is RpLS. Also in a
preferred
embodiment of the invention, the housekeeping gene probe is attached to one, 3
or 4
spots in the grid, and a random pool of 70mer oligonucleotides is attached to
3 spots in
the grid. The series of concentrations of external control probe is also
duplicated across
the grid spanning all its area. In a more preferred embodiment, the
housekeeping gene
is RpLS.
The process of the invention is even further characterized in that it
comprises the
step of:
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n) attaching a plurality of sample probes in other discrete spots of each said
grid as can
also be appreciated from figures 2A, 2B and 2C.
The process of the invention is further characterized by that the
hybridization
mixture comprises a plurality of sample targets to be hybridized on the array.
The
hybridization, the measurement of the level of hybridization and the
determination of the
amount of sample target present in the hybridizing mixture are done according
to
methods well known by a person in the art.
According to a preferred embodiment of the invention, and as can be seen in
figure 3, the external control probe and the sample probes are printed on the
solid
support, and more preferably by a Virtek ChipWriterPro~ (BioRad). According to
another
preferred embodiment of the invention, the Virtek ChipWriterPro~ has 12 pins,
and as
can be seen in figure 1, each pin produces a distinct grid with x rows and y
columns. In a
preferred embodiment of the invention, each grid can be 12 x 12, 12 X 13 or 12
x 14.
The 12 distinct grids printed by each pin represent a supergrid cluster. The
supergrid
cluster is then replicated across the surface of the glass slide to produce
the final
microarray. In a preferred embodiment of the invention, the supergrid is
replicated 4
times as can be appreciated in figures 1 and 2C.
The present invention also provides for a kit comprising:
a solid support having at least one grid;
at least one external control probe, said at least one external control probe
being
attached to at least one grid of said solid support in a series of
concentrations, said
series of concentrations being randomized in said at least one grid;
a hybridizing mixture containing at least one external control target being
complementary to said at least one external control probe; and
a plurality of sample probes attached to said solid support at discrete
locations.
The kit is further characterized in that the hybridizing mixture further
comprises a
plurality of sample targets to be hybridized on the array.
In a preferred embodiment of the invention the series of concentrations of the
external control probe of the kit varies from 0.0015 to 100 pM. and more
preferably it is
0.0015, 0.0031, 0.0061, 0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78,
1.5625,
3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 NM.
17
CA 02485055 2005-06-23
In a preferred embodiment of the invention the external control probe and the
sample probes of the kit of the invention are chosen from DNA, RNA,
oligonucleotides,
cDNA, proteins and antibodies and more preferably the external control probe
and the
sample probes of the kit are oligonucleotides, the oligonucleotides are even
more
preferably 70mer oligonucleotides. In a preferred embodiment of the invention
70mer
oligonucleotides of the kit are designed and synthesized according to the
following
characteristics:
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
- less than 20% cross homology; and/or
- less than 20 contiguous bases in common with top BLAST hit gene.
In another preferred embodiment of the invention the 70mer oligonucleotides of
the kit are attached to the solid support at a concentration chosen from 60,
40, 30, 15,
7.5 and 3.5 NM and more preferably at 40NM.
Examples
The following examples are illustrative of the applicability of the present
invention
and are not intended to limit its scope. Modifications and variations can be
made therein
without departing from the spirit and scope of the invention. Although any
method and
material similar or equivalent to those described herein can be used in the
practice for
testing of the present invention, the preferred methods and materials are
described. The
following experimental procedures and materials were used for the examples set
fort
below in the normalization of the inventors ToxArray~.
A) Materials And Methods
1. Gene selection and probe design
Genes predictive of toxicant exposure were established from in house gene
expression data, gene expression studies in the current literature, and
statistical re-
18
CA 02485055 2005-06-23
analysis of publicly available microarray data.
The following information was acquired through publicly available databases
for each
gene:
~ Official gene symbol and name
~ Gene sequence (RefSeq or GenBank accession number)
~ UniGene Cluster ID
~ Alternate gene names and aliases
~ Ontology
~ LocusLink ID
~ Ensemble Gene ID
~ Ensembl Transcript ID
The current list consists of 1100 genes. Figure 4 displays the gene list
grouped
by ontology.
Custom 70mer oligonucleotides were designed and synthesized for each gene by
Qiagen according to the following criteria:
- less than 60% GC content
- less than 1000 bases from the 3' end
- Longest stem less than 9 bases
- less than 70% cross homology
- less than 20 contiguous bases in common with top BLAST hit gene.
2. Oligonucleotide test set
Prior to printing the high density array, a set of control oligos provided by
Qiagen
were used to optimize array printing and hybridization protocols. These
comprise:
- Positive control oligos designed for common housekeeping genes (Table 1 ).
- Random 70mer oligos designed to have no cross homology to any known
mouse genes.
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CA 02485055 2005-06-23
Table 1
IIIV u~~
~i,,, ~, y~~ ,
Ldh1 ~i,e
Lactate dehydrogenase 1, A chain
Hsp70-3Heat shock protein, 70 kDa 3
Gapd Glyceraldehyde -3-phosphate
dehydrogenase
Ubb Ubiquitin B
Tcea1 Transcription elongation factor
A (SII) 1
Eif4a2 Eukaryotic translation initiation
factor 4A2
Cyc1 Cytochrome c -1
Tcfe2a Transcription factor E2a
Nup62 Nucleoporin 62
RplS Ribosomal protein L5
Top1 Topoisomerase (DNA) I
Hsp84-1Heat shock protein, 84 kDa 1
In addition to the test oligos provided by Qiagen, buffer-only and random
hexamers
were also printed on our initial microarrays as negative control features.
3. in house microarray printing
Microarray printing was performed by the Virtek ChipWriterPro (BioRad). An
example
of printing a microarray using 12 printing pins is represented in figure 3.
4. Target labeling and hybridization protocols
Universal mouse reference RNA (Stratagene) was used for all target labelling
and
array hybridizations. All array experiments were carried out in two colours,
Cy5 and Cy3,
using the cRNA linear amplification and fluorescent labelling kit from Agilent
Technologies. All arrays were processed in an automated hybridization station
(Tecan),
images were obtained with ScanArray Express~ confocal laser scanner (Packard
BioSystems), and image analysis was performed by QuantArray~ image analysis
software (Packard Biosystems).
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CA 02485055 2005-06-23
B) Results
1. Glass slide and oligo printing buffer selection
The PowerMatrix~ and QMT Epoxy~ slides produced significantly lower background
signals compared to all other slides tested (data not shown). The PowerMatrix~
slide
was chosen over the QMT Epoxy~ slide because of its ability to detect more of
the test
oligo spots (Figure 5).
Oligo spotting buffer had a significant effect on spot intensity:
ArrayIT~ spotting solution produced spots with the highest signal intensities
while PBS
and Quantifoil I~ spotting solutions produced spots with the lowest signal
intensities
(Figure 6). ArrayIT~ also produces the best quality spots based on spot size
and
morphology (Figure 7).
Tables 2 and 3 list respectively the glass slides and the oligo printing
buffers tested.
Table 2
~~~ ~ ' ~~ ~,~ ~ ' , ~~ ' ~
r~3t~ ~ ' I~~t'~~'~~G~Ut'~1'~~
~~'~'1~
, .,
, ~
j 3 ~~L fi '.
L~
x ~ AQ
.,.
Ev.p~.
, 3
~" - f" ~~k
QMT Epoxy Quantifoil Micro Tools
GmbH
PowerMatrix Full Moon Biosystems
SuperChip epoxysilane Erie Scientific
SuperAldehyde Telechem International
CodeLink Activated Amersham Biosciences
SpotOn Scandinavian Micro Biodevices
Table 3
~~ 1~~1~1f~~ i~~ ~ ~~~~~~~~'
~Ir
~ ~ .
r
ArrayIT Telechem International
Spotting Solution Quantifoil Micro Tools
I GmbH
Spotting Solution Quantifoil Micro Tools
II GmbH
Phosphate Buffered In house
Saline
(PBS)
21
CA 02485055 2005-06-23
2. Optimization of oligo printing concentration
To determine the optimal printing concentration, the oiigo test set was
printed at 60,
40, 30, 15, 7.5, and 3.75 pM.
- Large intensity gains are apparent when the oligo printing concentration is
increased to 40 wM (Figure 8).
- Intensity gains were minimal for printing concentrations > 40 pM (Figure 8).
The optimal oligo printing concentration was determined to be 40 ~.M, Figure 9
displays a typical microarray image of the oligo test set printed under
optimal oligo
printing concentration, printing buffer, and glass slide.
3. Microarray normalization feature
The majority of microarray analyses rely on normalization methods that assume
evenly distributed changes and/or absence of global shifts using microarray
features
such as housekeeping genes, spotted microarray sample pools, genomic DNA, or
all
genes.
Normalization based on the above features are not appropriate for the
inventors
ToxArray~ because global unbalanced changes are expected due to its relatively
small
size and the fact That genes were chosen based on evidence of transcript level
changes
as a result of toxicant exposure.
The use of external control features, made up of a non-murine oligonucleotide
and
specific RNA, allows a better comparison between slides and overcomes the
problem of
global changes in gene expression.
In addition to their use in normalization, external control features can also
be used to
cope with local, intensity-dependent systematic variation by representation in
sufficient
numbers in each grid and to monitor sample labelling and optimization of
microarray
hybridization protocols.
4. External control feature design
The inventors have opted to add a constant quantity of external RNA to each
total
RNA sample while varying the amounts of external control oligo printed on the
array
(Figure 10).
An oligo probe was designed for the Arabidopsis thaliana chlorophyll synthase
gene.
External control target RNA was prepared through in vitro transcription of A.
thaliana
chlorophyll synthase cDNA present in a plasmid provided by the Ontario Cancer
22
CA 02485055 2005-06-23
Institute. The chlorophyll synthase target RNA is added into the mouse target
RNA,
labeled with a fluorescent dye, and hybridized to the array. All methods
followed are
methods known in the art.
External control features of different concentrations will produce spots with
different
intensities as a result of hybridization of the external target RNA to the
oligo content in
the spot.
5. Optimization of external control oligo dilution series
Microarrays were printed with external control oligo dilutions ranging from
100 ~,M to
0.0015 wM. External control RNA was spiked into reference RNA (Stratagene)
prior to
labelling. 2ng, 8ng, 18ng, 20ng, and 40ng external control RNA spikes were
tested. A
series of 19 external control dilutions was found to be optimal, covering the
complete
range of expression levels observed for the test oligo set. (Figure 11 ) and
reproducible
across experiments (Figure 12).
6. Microarray feature printing design
Systematic en-ors arising reproducibly as a result of experimental procedure
contribute to inaccuracies in measured gene expression levels.
The spatial arrangement of features on a microarray resulting from printing
design is
one of the major sources of systematic error:
- In Figure 13 the spatial periodicity of spot intensity based on array grid
position
is evident.
- When the same data is sorted by printing pin (Figure 14), a clear difference
in
spot intensity for the same feature is observed based on printing pin.
The ToxArray~ has been designed such that all control and external
normalization features are printed by each pin resulting in their presence in
every grid on
the array.
Each grid on the ToxArray~ is printed in quadruplicate over the array area to
account for within-slide spatial variations.
Each grid consists of 96 unique gene features and an identical layout of
control
features (Figure 1) including:
- external control dilutions
- RpL5 housekeeping gene
- negative controls (buffer-only and randomized 70mers).
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CA 02485055 2005-06-23
7. Quality test
As can be appreciated from figure 2, by starting at extremely low molarity and
going up to saturation, the inventors span the linear dynamic range of the
signal
intensities, allowing the choice of one or several spots to use within each
subgrid for
normalizing.
Figure 15 shows the result from a recent quality test of the inventors
TOXARRAY~ plotting an external control dilution series, ensuring that within a
subgrid
one is spaning the range of signal intensities from background to saturation.
This allows
to choose an appropriate spot or two within each subgrid to normalize grids
from top to
bottom. As can be appreciated from figure 16, the system can thus can be used
to
simply detect differential hybridization (increasing grid number is going down
the chip - if
the line is sloping down or up, it indicates signal intensity of the same spot
differs from
top to bottom of the chip).
The inventors have thus designed an oligonucleotide-based microarray
representing more than 1100 genes predictive of toxicant exposure based on
gene
expression data in current literature, in house data, and statistical re-
analysis of relevant
existing microarray data in which an external control feature for
normalization has been
designed and tested for its ability to mimic the range of observed expression
levels for a
test set of oligos. The individual grid layout and number of control and
external
normalization features per grid have been designed to cope with sources of
both
systematic error and spatial variation.
24
CA 02485055 2005-06-23
Claims
1- An array system comprising:
a solid support having at least one grid;
at least one external control probe, said at least one external control probe
being
attached to at least one grid of said solid support in a series of
concentrations, said
series of concentrations being randomized in said at least one grid;
a hybridizing mixture containing at least one external control target being
complementary to said at least one external control probe; and
a plurality of sample probes attached to said solid support at discrete
locations.
2- The array system according to claim 1, wherein the hybridizing mixture
further
comprises a plurality of sample targets to be hybridized on said array.
3- The array system according to claim 1, wherein the at least one external
control probe
and the sample probes are chosen from DNA, RNA, oligonucleotides, cDNA,
proteins
and antibodies.
4- The array system according to claim 1, wherein said series of
concentrations of the at
least one external control probe varies from 0.0015 to 100 NM.
5- The array system according to claim 4, wherein said series of
concentrations of the at
least one external control probe is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244,
0.0488,
0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100
NM.
6- The array system according to claim 1, wherein the sample probes are
oligonucleotides.
7- The array system according to claim 6, wherein the oligonucleotides are
70mer
oligonucleotides.
8- The array system according to claim 7, wherein the 70mer oligonucleotides
are
designed and synthesized according to the following characteristics:
CA 02485055 2005-06-23
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
- less than 20% cross homology; and/or
- less than 20 contiguous bases in common with top BLAST hit gene.
9- The array system according to claim 8, wherein the 70mer oligonucleotides
are
attached to the solid support at a concentration chosen from 60, 40, 30, 15,
7.5 and 3.5
NM.
10- The array system according to claim 9, wherein the 70mer oligonucleotides
are
attached to the solid support at a concentration of 40NM.
11- The array system according to claim 1, wherein it is a microarray system.
12- The array system according to claim 1, wherein said at least one external
control
probe and said plurality of sample probes are printed on said solid support.
13- A process of normalizing an array system, comprising the steps of:
a) providing a solid support comprising at least one grid;
b) attaching at least one external control probe to said solid support in a
series of
concentrations, each of said series of concentration of said at least one
external control
probe being attached to a grid of said solid support at a different discrete
location from
another concentration of said series, said series of concentrations being
randomized in
said grid;
c) attaching at least one sample probe on at least one grid to said solid
support;
d) making several grids by repeating steps a) and b);
e) hybridizing the hybridizing mixture to the microarray;
f) measuring level of hybridization between the at least one external control
probe and
the at least one external control target and between the plurality of sample
probes and
plurality of sample targets; and
g) determining amount of sample target present in the hybridizing mixture.
14- The process according to claim 13, further comprising the following step:
26
CA 02485055 2005-06-23
h) attaching the at least one external control probe series of concentration
in duplicate
on said grid;
15- The process according to claim 13, further comprising at least one of the
following
steps:
i) adding a buffer only to at least a discrete spot on said grid;
j) leaving at least one discrete spot in said grid empty;
k) attaching a random hexamer to one spot on said grid;
I) attaching a random pool of 70mer oligonucleotides to at least one discrete
spot on said
grid; and
m) attaching a probe from a house keeping gene to at least one discrete spot
on said
grid.
16- The process according to claim 15, wherein the house keeping gene is RpLS.
17- The process of normalization according to claim 15, further comprising the
step of:
n) attaching a plurality of sample probes in other discrete spots of each said
grid.
18- The process of normalization according to claim 15, wherein the
hybridization
mixture comprises a plurality of sample targets to be hybridized on the array.
19- The process of normalization according to claim 18, wherein the at least
one
external control probe and of the sample probes are chosen from DNA, RNA,
oligonucleotides, cDNA, proteins and antibodies.
20- The process of normalization according to claim 19, wherein said series of
concentrations of the at least one external control probe varies from 0.0015
to 100 NM.
21- The process of normalization according to claim 20, wherein said series of
concentrations of the at least one external control probe is 0.0015, 0.0031,
0.0061,
0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5,
25, 50, 60,
80 and 100 pM.
27
CA 02485055 2005-06-23
22- The process of normalization according to claim 21, wherein the sample
probes are
oligonucleotides.
23- The process of normalization according to claim 22, wherein the
oligonucleotides are
70mer oligonucleotides.
24- The process of normalization according to claim 23, wherein the 70mer
oligonucleotides are designed and synthesized according to the following
characteristics:
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
- less than 20% cross homology; and/or
- less than 20 contiguous bases in common with top BLAST hit gene.
25- The process of normalization according to claim 24, wherein the 70mer
oligonucleotides are attached to the solid support at a concentration chosen
from 60, 40,
30, 15, 7.5 and 3.5 NM.
26- The process of normalization according to claim 25, wherein the 70mer
ofigonucleotides are attached to the solid support at a concentration of 40NM.
27- A kit, comprising:
a solid support having at least one grid;
at least one external control probe, said at least one external control probe
being
attached to at least one grid of said solid support in a series of
concentrations, said
series of concentrations being randomized in said at least one grid;
a hybridizing mixture containing at least one external control target being
complementary to said at least one external control probe; and
a plurality of sample probes attached to said solid support at discrete
locations.
28- The kit according to claim 27, wherein the hybridizing mixture further
comprises a
plurality of sample targets to be hybridized on said array.
28
CA 02485055 2005-06-23
29- The kit according to claim 28, wherein the at least one external control
probe and the
sample probes are chosen from DNA, RNA, oligonucleotides, cDNA, proteins and
antibodies.
30- The kit according to claim 29, wherein said series of concentrations of
the at least
one external control probe varies from 0.0015 to 100 pM.
31- The kit according to claim 30, wherein said series of concentrations of
the at least
one external control probe is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244, 0.0488,
0.0077,
0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 NM.
32- The kit according to claim 31, wherein the sample probes are
oligonucleotides.
33- The kit according to claim 32, wherein the oligonucleotides are 70mer
oligonucleotides.
34- The kit according to claim 33, wherein the 70mer oligonucleotides are
designed and
synthesized according to the following characteristics:
- less than 60% G C content
- less than 1000 basis from the 3' end;
- longest stem being less than 9 bases;
- less than 20% cross homology; andlor
- less than 20 contiguous bases in common with top BLAST hit gene.
35- The kit according to claim 33, wherein the 70mer oligonucleotides are
attached to
the solid support at a concentration chosen from 60, 40, 30, 15, 7.5 and 3.5
pM.
36- The kit according to claim 35, wherein the 70mer oligonucleotides are
attached to
the solid support at a concentration of 40NM.
29
