U.S. patent application number 10/865475 was filed with the patent office on 2004-12-30 for microchip arrays of regulatory genes.
This patent application is currently assigned to Sir Mortimer B. Davis - Jewish General Hospital. Invention is credited to Wang, Eugenia.
Application Number | 20040265886 10/865475 |
Document ID | / |
Family ID | 22715426 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265886 |
Kind Code |
A1 |
Wang, Eugenia |
December 30, 2004 |
Microchip arrays of regulatory genes
Abstract
Microarray technology is a fast-growing field of biomedical
research, aiming to investigate changes in molecular features of
hundreds of genes. The multiple parallel processing of information
generated from matrices of huge numbers of loci on a solid
substrate has allowed the gathering of gene signatures defining
specific biological states. A new approach has been developed to
facilitate this process wherein genes of the same regulatory
modality are selected. The transcriptional regulation of these
genes is related to the same control element, the E-box, defined by
the sequence CACGTG. PCR products of selected regions of all known
genes either binding to this sequence or whose expression is
dependent on this binding, as well as genes interacting with
E-box-binding genes and control genes, are arrayed on a nylon
membrane or other appropriate microchip susbstrate, which is then
used as an E-box-specific microarray. The transcriptionally
regulated profile of E-box-related genes specific to a given
cultured cell sample is then determined by unique labeled cDNAs
probes produced from RNAs isolated from the culture of
interest.
Inventors: |
Wang, Eugenia; (Prospect,
KY) |
Correspondence
Address: |
PATREA L. PABST
PABST PATENT GROUP LLP
400 COLONY SQUARE
SUITE 1200
ATLANTA
GA
30361
US
|
Assignee: |
Sir Mortimer B. Davis - Jewish
General Hospital
Quebec
CA
|
Family ID: |
22715426 |
Appl. No.: |
10/865475 |
Filed: |
June 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10865475 |
Jun 10, 2004 |
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09821203 |
Mar 29, 2001 |
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6759197 |
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60193888 |
Mar 31, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/6.1; 435/6.18; 506/4; 536/24.3 |
Current CPC
Class: |
C12Q 2527/107 20130101;
C12Q 2565/501 20130101; C12Q 2565/501 20130101; B01J 2219/00722
20130101; C12Q 1/6809 20130101; C12Q 1/6883 20130101; C12Q 1/6809
20130101; C12Q 2600/158 20130101; C12Q 1/6809 20130101; C40B 40/06
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04; C12M 001/34 |
Goverment Interests
[0002] The United States government has certain rights in this
invention by virtue of grants to Eugenia Wang from the National
Institute on Aging (AG09278) and from the Defense Advance Research
Project Agency (DARPA) of the Department of Defense of the United
States of America.
Claims
1. An array of genes each comprising an E-box regulatory sequence
in its promoter, interacting with a gene binding to the E-box
regulatory sequence or whose expression is dependent on this
binding.
2. The array of claim 1 wherein the genes further comprise
sequences encoding proteins associated with a particular state,
disease or disorder.
3. The array of claim 2 wherein the state is age.
4. The array of claim 3 wherein the genes are isolated from a young
animal.
5. The array of claim 3 wherein the genes are isolated from an old
animal.
6. The array of claim 2 further comprising housekeeping genes whose
expression does not change significantly as the state changes.
7. The array of claim 6 comprising at least nine housekeeping
genes.
8. The array of claim 6 wherein the state is age and the expression
of the housekeeping genes does not change as the animals age.
9. The array of claim 8 wherein the housekeeping genes are selected
from the group consisting of tyrosine 3-monooxygenase/tryptophan
5-monooxygenase activation protein, hypoxanthine
phosphoribosyltransferas- e I (Lesh-Nyhan syndrome), Major
histocompatibility complex, class I, C, Ubiquitin C,
Glyceraldehyde-3-phosphate dehydrogenase, Human mRNA fragment
encoding cytoplasmic actin, 60S Ribosomal protein L13A, and
Aldolase C.
10. The array of claim 1 wherein the genes are present in nanomolar
quantities.
11. A set of primers for use in detecting changes in expression of
genes comprising an E-box regulatory sequence in its promoter or
interacting with a gene binding to the E-box regulatory sequence,
having a length between 480 and 700 base pairs length and a melting
point between 75 and 85.degree. C., wherein the primers include
non-consensus sequence with protein coding sequence so that there
is no detectable hybridization between homologous genes.
12. The set of primers of claim 11 where there is no hybridization
between homologous genes.
13. The set of primers of claim 11 wherein the primers do not
hybridize to homologous genes having the same degree of homology
and c-myc and c-myc associated genes.
14. The set of primers of claim 11 comprising a label.
15. The set of primers of claim 14 wherein the label is selected
from the group consisting of digoxigenine label, radiolabels and
fluorescent labels.
16. A kit for detecting changes in expression of genes which is
associated with a particular state, disease or disorder comprising
a) an array of genes each comprising an E-box regulatory sequence
in its promoter or interacting with a gene binding to the E-box
regulatory sequence and one or more housekeeping genes; b) a set of
E-box primers for use in detecting changes in expression of the
genes in the array; and c) means for detecting hybridization
between the primers and the regulatory sequences.
17. The kit of claim 16 wherein the array of genes is prepared from
cells or tissues of an animal characterized by a particular state,
disease or disorder.
18. The kit of claim 17 wherein the animal is selected based on age
or a disease or disorder associated with cancer, the neuronal
system, the musculoskeletal system, or cardiovascular system.
19. The kit of claim 17 further comprising means for quantitating
the amount of expression.
20. The kit of claim 18 wherein the animal or cells from which the
genes are derived have been treated with one or more compounds or
dosage regimes to screen for an effect of the compound or dosage
regime on the state, disease or disorder.
21-29. (canceled)
Description
[0001] This application claims priority to U.S. Ser. No. 60/193,888
filed Mar. 31, 2000.
BACKGROUND OF THE INVENTION
[0003] With the advent of the Human Genome Project, one is
confronted with voluminous information demonstrating that
biological systems may be controlled by hundreds of genes working
in concert. A single glance at the ever-increasing number of genes
involved in signal transduction makes one wonder just how many
genes are needed to choreograph the symphonic dance of implementing
a signal, from the receptor-ligand binding to the nuclear response
of transcriptional activation. During the 1980's and early 1990's,
biologists were busy dissecting single genes' functions from the
reductionist point of view. This approach, while thorough in its
exact methodological analysis of genetic impact, lacks the expanded
vision of how each particular single gene functions in the context
of many sister genes or partners, to accomplish a biological task.
Thus, it is not surprising that the technology of high-throughput
gene screening is emerging rapidly, in the attempt to identify tens
or hundreds of genes whose changes, viewed in composite genetic
signatures, define a particular physiological state. This gene
signature approach, complemented by single gene analysis, provides
a vertical, in-depth analysis of an individual gene's function, as
well as the comprehensive picture of the pattern of gene expression
in which the particular gene functions. The notion of genetic
signature can be further generalized to address the question of
inter-individual variance, by comparing individuals from cohorts of
hundreds or thousands.
[0004] The unfathomable task of comparing several dozens of single
nucleotide polymorphisms (SnP) in a hundred people can now be
approached easily by DNA biochip technology (Wang, et al. Science
280:1077-1082 (1998)). For example, a p53 DNA chip is used
popularly for the identification and gene screening of unique
cancer risks, to discover new SnPs as well as screening known SnPs.
Either task needs a fast, multiplex approach requiring data entry
on the scale of hundreds and thousands, a demand that can only be
met by high-throughput technology. The presently available
microarray biochip technology is certainly the method of choice to
solve the problem of complexity, and the previously impossible task
of defining a genetic signature for a unique person in a cohort
with accuracy and speed that are impossible by the conventional
diagnostic approach. Therefore, from bedside researchers to bedside
physicians, there is intense interest in the technology of
microarray analysis, for screening or identifying tens or hundreds
of genes related to disease or normal states of a given person or
biological system.
[0005] cDNA and oligonucleotide microarrays are becoming an
increasingly powerful technique for investigating gene expression
patterns. In spite of the fast progress in this field, some
limitations of the technique persist. One of the major obstacles is
the requirement for a large amount of mRNA. Another problem with
existing microarray systems is data mining; while information on
expression of tens of thousands genes is absolutely vital to
estimate the functions of new genes, in some instances a researcher
is interested in the expression profile of only a subset of genes,
in many physiological conditions.
[0006] It is an object of the present invention to provide a method
and materials for the rapid analysis of genetic information based
on a common regulatory feature.
[0007] It is a further object of the present invention to provide a
method and materials for sensitive and quick analysis of genetic
information present in very small quantities.
SUMMARY OF THE INVENTION
[0008] Microarray technology is a fast-growing field of biomedical
research, aiming to investigate changes in molecular features of
hundreds of genes. The multiple parallel processing of information
generated from matrices of huge numbers of loci on a solid
substrate has allowed the gathering of gene signatures defining
specific biological states. A new approach has been developed to
facilitate this process wherein genes of the same regulatory
modality are selected. The transcriptional regulation of these
genes is related to the same control element, the E-box, defined by
the sequence CACGTG. PCR products of selected regions of all known
genes either binding to this sequence or whose expression is
dependent on this binding, as well as genes interacting with
E-box-binding genes and control genes, are arrayed on a nylon
membrane or other appropriate microchip susbstrate, which is then
used as an E-box-specific microarray. The transcriptionally
regulated profile of E-box-related genes specific to a given
cultured cell sample is then determined by unique labeled cDNAs
probes produced from RNAs isolated from the culture of
interest.
[0009] The production of E-box microarrays provides an approach to
custom-adapt the gene screening task to analyze a subgroup of gene
expressions controlled by the same molecular modality. E-box
binding-related genes represent a specific group of basic
helix-loop-helix/leucine zipper transcription factors, recognizing
the core-binding site CACGTG. They play important roles in
regulation of basic cellular functions, like proliferation and
apoptosis (c-Myc) or tissue-specific differentiation (Myod). As
demonstrated by the example, careful selection of genes for the
microarray allowed extraction of E-box gene specific signatures of
HeLa cells and normal human lymphocytes. The significant
differences in expression of 3-6 genes out of 61 are already much
more manageable than can be detected from ordinary microarrays with
massive numbers of genes, in the hundreds or thousands.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 depicts cDNA microarray hybridization for evaluation
of E-box binding-related gene expression. The matrix position, with
each gene's abbreviation, is written underneath each locus of three
repeats of dots with identical amounts deposited; the X-coordinates
denote the number 1, 2, 3, 4, and 5 positions, and Y-coordinates
denote the "a" through "o" positions. The matrix location for each
gene triplet is then identified as X, Y coordinates. For example,
5k denotes the position of N-Myc, and 3d denotes of the position of
Mad. The same coordinates are also included in Table 2.
[0011] FIGS. 2A and 2B shows the expression profiles of E-box
binding-related gene expressions in Hela cells. FIG. 2A--total RNA
was labeled with digoxigenin in RT reaction with gene specific
primers; FIG. 2B--mRNA was labeled with digoxigenin in RT reaction
with oligo(dT) primers. Arrows within the matrix show positions of:
I-Hela DNA (positive control); II-lambda DNA (negative control);
III-UBC; IV-RPL-13A; V-MBP-1; VI-HPRT1. The distance between dots
can be measured by the bar of 1 mm.
[0012] FIG. 3 depicts hybridization of products of multiplex PCR
with 5 pair of primers with cDNA microarray. Arrows within the
matrix point to: I-Mrdb; II-c-Myc p64; III-TFII-1; IV-ODC1;
V-cdc25A; VI-Hela genomic DNA.
[0013] FIG. 4 shows the relationship between concentrations of 5
genes including Mrdb, c-Myc, TFII-1, ODC1, and cdc25a, and
intensity of hybridization signals. Logarithmic approximation is
shown. Dot intensity is represented by the arbitrary units on the
Y-axis; concentration is measured as ng/ml on the X-axis.
[0014] FIGS. 5A and 5B show the expression profiles of
E-box-related genes in Hela cells (FIG. 5A), and normal human
lymphocytes (FIG. 5). Arrows within the matrix show positions of:
I-Aldolase C; II-Mad4; III-MBP-1.
[0015] FIGS. 6A. 6B and 6C are pairwise comparisons of E-box gene
expression in Hela cells and human lymphocytes. Two independent
hybridizations are averaged for each type of cell. FIG.
6A--Three-dimensional, and FIG. 6B--two-dimensional,
representations of differences in gene expression. Each panel
corresponds to one column in FIG. 5A, and each bar represents an
individual gene. FIG. 6C--Distribution of genes with common gain
(red) or loss (blue) of expression in dependence on relative ratio
value. The relative fold ratio between samples S1 and S2 is
computed as 1 R DM ( S 1 , S 2 ) = ( S 2 - S 1 ) max ( S 1 , S 2
)
[0016] which yields a value in the range of [-1,+1]. Positive
values correspond to up-regulation, and negative values correspond
to down-regulation, of genes in sample S2. The relative fold ratio
has a similar meaning to that of conventional fold ratio, except
that the value is normalized and symmetric, with clear physical
interpretation. R.sub.DM(S.sub.1,S.sub.2)=.+-.0.5 corresponds to a
two-fold up- or down-regulation in normalizing the two samples; a
set of housekeeping genes of relatively constant expression levels
were selected as controls, and linear normalization was
applied.
DETAILED DESCRIPTION OF THE INVENTION
[0017] E-box Regulatory Genes
[0018] The production of E-box microarrays provides an approach to
custom-adapt the gene screening task to analyze a subgroup of gene
expressions controlled by the same molecular modality. E-box
bindinig-related genes represent a specific group of basic
helix-loop-helix/leucine zipper transcription factors, recognizing
the core-binding site CACGTG. As used herein, E-box genes refer to
all genes having the E-box in their promoter region, as well as
E-box binding and interacting genes. They play important roles in
regulation of basic cellular functions, like proliferation and
apoptosis (c-Myc) or tissue-specific differentiation (Myod). As
demonstrated by the example, careful selection of genes for the
microarray allowed extraction of E-box gene specific signatures of
HeLa cells and normal human lymphocytes. The significant
differences in expression of 3-6 genes out of 61 are already much
more manageable than can be detected from ordinary microarrays with
massive numbers of genes, in the hundreds or thousands. For
example, in SAGA analysis of 45,000 genes, it was found that about
only 1% are differentially expressed in normal and cancerous human
cells (Zhang, et al., Science 276, 1268-1272 (1997)). A similar
estimation resulted from analysis of expression profiles in young
and old mice; expressions of only 1.8% of about 6,000 genes are
changed more than 2-fold (Lee, et al., Science 285, 1390-1393
(1999)).
[0019] The best-known representative of E-box-binding, genes is
c-Myc, whose transactivating activity plays crucial roles in the
regulation of cell cycle, proliferation and apoptosis (Eilers, Mol.
Cells 9, 1-6 (1999); Dang, C. V. Mol. Cell Biol. 19, 1-11 (1999);
Facchini and Penn, FASEB J. 12, 633-651 (1998)). For this reason,
genes interacting with or regulating expression for c-Myc, as well
as some target genes whose expression is E-box-binding-dependent,
are included in this microarray. Representative E-box genes are
shown in Table 2.
[0020] Housekeeping Genes
[0021] Housekeeping genes are used to normalize results of
expression. These are genes that are selected based on the
relatively invariable levels of expression in the system which is
being examined, for example, the state such as age or a particular
disease. Representative housekeeping genes are shown in Table 2.
These include tyrosine 3-monooxygenase/tryptophan 5-monooxygenase
activation protein, hypoxanthine phosphoribosyltransferase I
(Lesh-Nyhan syndrome), Major histocompatibility complex, class I,
C, Ubiquitin C, Glyceraldehyde-3-phosphate dehydrogenase, Human
mRNA fragment encoding cytoplasmic actin, 60S Ribosomal protein
L13A, and Aldolase C.
[0022] Probes
[0023] In the preferred embodiment, a set of primers for use in
detecting changes in expression of genes include the E-box
regulatory sequence, are between 480 and 700 base pairs length,
have a melting point between 75 and 85.degree. C. and include
non-consensus sequence with protein coding sequence, so that there
is no detectable hybridization between homologous genes, more
preferably where there is no hybridization between homologous
genes. Examples of homologous genes include c-myc and c-myc
associated genes.
[0024] Diseases and States
[0025] The changes in expression of the E-box regulatory genes
described herein can be used to assess changes associated with a
particular state or disease. The association of certain E-box genes
such as c-myc with cancer and neurodegeneration, and its role in
apoptosis, are well established. Other genes include yyl, myc-L1,
and myc-L2, which affect cells, cell components, and specific
molecules, for example, cardiomysin, myotube, osteoblasts, and
osteoclasts. Changes in expression of individual genes, either by
turning expression on or off, or altering the amount of expression,
can be used to assess changes in states such as age or diseases
associated with cancer of tissues such as breast, prostate, and
colon, immunological changes such as inflammation,
neurodegenerative diseases, cardiovascular disorders, and
musculoskeletal disorders, including disorders and diseases of
bones such as osteoarthritis and osteoporosis, and muscle
degeneration.
[0026] Screening
[0027] The arrays can be tested by screening with labeled probes to
determine if there is expression of a particular gene in the array
and how much, to thereby construct a "fingerprint" of the disease
or disorder at that time, using genes present in cells or tissues
obtained from one or more individuals having the disease or
disorder or characterized by a particular state, such as age. The
effect of a compound or composition on the disorder or disease or
state can also be assessed by comparing the fingerprint obtained
with control cells or tissues, and cells or tissues treated with
the compound or obtained from an animal treated with the compound
(or compounds, or dosage regime, or exposed to particular
conditions). This is especially useful for initial screening of the
effect of potential drugs, either to determine potential efficacy
and/or toxicity. Those compounds which appear promising can then be
further screened to determine if they can reduce or reverse the
severity of the disease or disorder. Compounds to be screened can
be proteins or peptides, sugars or polysaccharides, nucleic acid
molecules, or synthetic molecules.
[0028] MicroChip Array Technology and Analysis
[0029] Information Resources
[0030] There are several DNA microchip technology reviews in the
literature (Bowtell, D. D. L. Nature Genetics Supplement 21:25-32
(1999); Constantine, and Harrington Life Science News 1:11-13
(1998); Ramsay, G Nature Biotechnology 16:40-44 (1998)), and
several good web sites detailing the apparatus and protocols used
by other laboratories, nothing in the literature offers a
description of a working arrangement to serve as a user-friendly
guide. Table 1 lists several good web sites for highly active
laboratories in DNA microchip technology, as well as several
sources of robotics systems and equipment, imaging software and
systems and vendors of robotic components.
[0031] The Microarrayer
[0032] A turnkey microarrayer can be purchased, with an enclosure
for temperature, humidity and air quality control; a system such as
the GeneMachines.TM. OmniGrid (San Carlos, Calif.) would be
sufficient. Alternatively, to save on the cost of a robotic system,
a microarrayer can be built in the laboratory. The Brown Laboratory
web site, for example, gives full details for component
specifications, mechanical drawings for machined parts, a list of
vendors, an assembly guide, and free microarrayer software.
[0033] Operation of the Tips, XYZ Motion Control, and Computer
Program
[0034] The robotic gantry of a typical printing tip microarryer is
composed of 3 individual assemblies of linear robotic tables, and
motors driven by 3 corresponding amplifiers which are coupled to a
motion controller in the driving computer. All of this forms the
appropriate 3-axis motion control system (i.e.: X, Y and Z axes)
for microarraying. The three perpendicular axes allow for sampling,
printing and washing with the components of the microarryer
system.
[0035] Printing Substrate and Samples
[0036] In terms of a printing substrate for producing the
microchips, poly-L-lysine-coated glass slides seem to work best to
immobilize the printed DNA. Nylon hybridization membranes can also
be used as the printing substrate, and allow for a much easier
immobilization protocol, as well as better visualization if a
calorimetric method is used for hybridization detection.
[0037] To contain the samples, conical 96-well microplates work
well by localizing small volumes of sample in the wells. When
printing many different samples, 384-well microplates are best due
to their higher capacity and low storage volume and the smaller
sample sizes (.ltoreq.10 .mu.l) can be used readily. During
storage, sample plates should be covered with an adhesive-backed
plastic seal, to prevent sample loss by evaporation.
[0038] Sample Preparation
[0039] Samples prepared for printing are loaded into 384-well
microplates, 10 .mu.l aliquots per well. These samples can be used
for up to 8 to 10 printing runs, with proper storage. In printing
arrays with the ArrayIt.TM. printing tips on the GeneMachines.TM.
OmniGrid microarrayer, it is possible to print several thousand
spots onto one chip either in one array or duplicate arrays on one
chip. The printing tip delivery volume is approximately 1 nl per
spot with a spot diameter of approximately 100 .mu.m. Therefore,
depending upon the surface area of the substrate being used as the
chip and the number of tips used for printing, several large arrays
are possible with close spacing (less than 100 um) for up to 100
chips per run. For typical experiments in this laboratory, arrays
are printed ill duplicate 20.times.20 arrays per chip with a spot
spacing of 250 .mu.m still using between 20 to 30 chips per
run.
[0040] To extend the lifetime of the samples, after printing, the
microtiter plates are sealed with adhesive-backed plastic covers in
addition to the microplate lids. Furthermore, before using the
stored samples again, the microplates are centrifuged to gather any
condensate in the wells, and to localize the sample fluids at the
bottom of each well.
[0041] Array Analyzer/Imaging System
[0042] Depending upon the selected approach to hybridization
analysis of the printed microarrays, a system fitted onto an
existing microscope, a microarray scanner or confocal laser scanner
may be purchased, or a con focal laser scanner may be built.
[0043] The system used to compile the digital microarray images is
built around an Olympus BH-2 upright light microscope, fitted with
a Sony color CCD camera, an Applied Scientific Instrumentation
(Eugene, Oreg.) X-Y scanning stage, and a fiber optic ring
illuminator from Edmund Scientific Co. (Barrington, N.J.). EMPIX
Imaging, Inc. (Mississauga, ON) assembled the system for compiling
microarry images, containing a 24 bit frame grabber; it is
installed in a 450 MHz P3 PC equipped with 512 Mb RAM and a 19"
SVGA monitor, where the image acquisition and system control are
governed under the Windows 98 operating system by Northern
Eclipse.TM. imaging software. A 3COM.TM. 10/100 Base TX network
card installed in the computer links the imaging computer to a
small LAN (Lynksys, Irvine, Calif.), containing a color laser
printer and two other computers used for image analysis and data
storage.
[0044] The size of the arrays and individual spots dictates the use
of low power objectives (either 2.5.times. or 4.times.) and the X-Y
scanning stage to capture the image of the entire array.
[0045] Many of our microarray experiments are done using nylon
membranes (Hybond-N) as the printing substrate. Probes are labeled
with DIG-dUTP in a reverse transcription reaction; target/probe
hybridization is detected with anti-DIG-coupled alkaline
phosphatase, and a subsequent reaction of the alkaline phosphatase
with an NBT/BCIP stain/substrate. This method requires the ring
illuminator to distinguish artifacts from array spots on the
stained hybridization membranes. Otherwise, if poly-L-lysine coated
glass slides are used as the microarray printing substrate,
illumination of the microarray specimen is carried out
normally.
[0046] Image Quantitation
[0047] When the microarray digital imaging routine is completed,
the compiled montage can be transferred by way of the network to
the computer stations devoted to image analysis and data storage.
The microarray images are created as TIFF files; before
quantitation can begin, the raw digital images are filtered to bear
only the microarray signal data, aligned in Adobe PhotoShop.TM.
software, and then transferred to the GeneAnalyzer microarray
analysis software. GeneAnalyzer removes the background, and the
reduced digital microarray images are passed through an image
location routine to optimally localize the spots of the microarray
image. When the GeneAnalyzer software has "grabbed" the individual
spots of the reduced digital microarray image, the program can
proceed to quantitate the density of the individual spots. Each
spot on the microarray is then regarded as an individual signal,
and its intensity serves as the foundation of the data needed to
reflect the hybridization reaction. After comparison with
appropriate positive and negative controls for nonspecific
reactions, true signal value is subtracted from noise to produce
the desired information on each hybridization reaction.
[0048] The microarray spot density data are transferred into an
analysis routine in the mathematical analysis software, MATLAB, for
graphical representation of all data; the density values, as well
as the respective calculated values, of all digitized microarray
data are tabulated in a Microsoft Excel.TM. spreadsheet. A full
record of the progression of images, tabulated data and all
graphical representations can immediately be printed to complete
the microarray experiment analysis.
[0049] Labels for Probes and Detection
[0050] Microarrays typically contain at separate sites nanomolar
(less than picogram) quantities of individual genes, cDNAs, or ESTs
on a substrate such as a nitrocellulose or silicon plate, or
photolithographically prepared glass substrate. The arrays are
hybridized to cDNA probes using standard techniques with
gene-specific primer mixes. The nucleic acid to be analyzed--the
target --is isolated, amplified and labeled, typically with a
fluorescent reporter group, radiolabel or phosphorous label probe.
After the hybridization reaction is completed, the array is
inserted into the scanner, where patterns of hybridization are
detected. The hybridization data are collected as light emitted
from the reporter groups already incorporated into the target,
which is now, bound to the probe array. Probes that perfectly match
the target generally produce stronger signals than those that have
mismatches. Since the sequence and position of each probe on the
array are known, by complementarity, the identity of the target
nucleic acid applied to the probe array can be determined.
[0051] There are a variety of labels that are used. cDNAs and ESTs
can be detected by autoradiography or phosphorimaginig (.sup.32 p).
Fluorescent dyes are also used, and are commercially available from
suppliers such as Clontech.
[0052] In the preferred embodiment the label is digoxigenin (DIG).
This specific enzymatic labeling probe allows the end result of
detecting hybridization reaction intensity by colorimetric
evaluation of alkaline phosphatase-coupled antibody to DIG. The
enzymatic deposit on each locus of the E-box microarray can be
readily analyzed by an upright microscope attached to a CCD camera,
without the problem of the long delay needed for exposure time with
radioactive probes, or the photobleaching and high background
reaction problem associated with the fluorescent probe
approach.
1TABLE 1 Informative web sites for DNA microarray technology URL
DNA microarray technology web sites Automation and Miniaturization
in Genome Analysis,
http://www.mpimg-berlin-dahlem.mpg.de/.about.autom/autom.htm Max
Plank Institute for Molecular Genetics Department of Molecular
Biotechnology, http://chroma.mbt.washington.edu/mod_www/ University
of Washington Functional Genomics Group,
http://sequence.aecom.yu.edu/bioinf/funcgenomic.html Albert
Einstein College of Medicine Genomics Group,
http://w95vcl.neuro.chop.edu/vcheunng Children's Hospital of
Philadelphia Laboratory of Cancer Genetics,
http://www.nhgri.nih.gov/Intramural_research/Lab_cancer/ National
Human Genome Research Institute Joint Genome Institute, http
:/llnl.gov/automation-robotics/poster.1.html Lawrence Livenmore
National Laboratory Pat Brown Laboratory, http://cmgm.stanford.edu-
/pbrown Stanford University Stanford DNA sequence and Technology
Center http://-sequence.stanford.edu/group/techdev/ Stanford
University Microarrayers, imaging systems and scanners Applied
Scientific Instrumentation, Inc. http://www.ASIimaging.com/ Axon
Instruments, Inc. http://axon.com/GN_Genomics.html Beecher
Instruments http://www.beecherinstruments.com/ BioDiscovery, Inc.
http://www.biodiscovery.com/ BioRobotics, Ltd.
http://www.biorobotics.com/ Empix Imaging, Inc.
http://www.empix.com/ GeneMachines, Genomic Instrumentation
Services, Inc. http://www.genemachines.com/ General Microarray
Information http://www.microarray.org/ General Scanning, Inc.
http://www.genscan.com/ Genetic MicroSystems, Inc.
http://www.geneticmicro.com/ Genometrix, Inc.
http://www.genometrix.com/ Genomic Solutions
http://www.genomicsolutions.com/ Imaging Research, Inc.
http://www.imagingresearch.com/ Intelligent Automation
http://www.ias.com Molecular Dynamics, Inc.
http://www.mdyn.com/arrays/arraywhat.htm Radius Biosciences
http://www ultranet.com/.about.radius Research Genetics
http://www.resgen.com ScanAlyze software http://bronzino.stanford.-
edu/ScanAlyze/ Telechem International, Inc.
http://www.wenet/.about- .telechem/ Western Technology Martketing
http://www.westerntechnolo- gy.com/ Robotics Galil
http://galilmc.com/ Parker-Compumotor http://www.compumotor.com/
Parker-Daedal http://www.daedalpositioning.com/
EXAMPLE 1
Digoxigenin Enzymatic Detection for Microarray Analysis of E-Box
Binding Related Gene Expression
[0053] Realizing the advantages and problems of cDNA microarrays
for expression profiling, in this study a new approach was
developed based on utilizing digoxigenin (DIG) to label target cDNA
produced from gene-specific primers, with subsequent incubation
with anti-digoxigenin antibody conjugated with alkaline phosphatase
(AP), and colorimetric or chemiluminescent detection. A set of
genes containing, the E-box binding element (CACGTG), located in
promoter regions of many genes, was selected as the probes.
Probably the best-known representative of E-box-bindinig genes is
c-Myc, whose transactivating activity plays crucial roles in the
regulation of cell cycle, proliferation and apoptosis (Eilers, M.
Mol. Cells 9, 1-6 (1999); Dang, C. V. Mol. Cell Biol. 19, 1-11
(1999); Facchini and Penn FASEB Journal 12,633-651 (1998)). Genes
interaction with or regulating expression for c-Myc, as well as
some target genes whose expression is E-box-bindinig-dependent, are
included in this microarray. These custom-designed microarrays,
combined with the enzymatic approach to label hybridization probes,
allow the development of an inexpensive, user-friendly system for
high-throughput gene screening assay of specific subgroups of gene
expressions.
[0054] Materials and Methods
[0055] Selection of Probes for Arraying
[0056] E-box-binding proteins, as well as c-Myc-regulating,
-interacting and target genes, were chosen from different data
bases--GeneAtlas (http://www.citi2.fr/GENATLAS), GeneCards
(http://bioinfo.weizmann.ac.il/- cards), GenBank
(http://www.ncbi.nkm.nih.gov/Web/Genbank) and PubMed
(http://www.ncbi.nlm.nih.gov/PubMed). Unigene
(http://www.ncbi.nlm.nih.go- v/UniGene/index.html) cluster numbers
and sequences were used to identify genes and verify their
uniqueness. Nine housekeeping genes, as well as HeLa cell DNA were
selected as positive controls; as negative controls, lambda DNA and
2.times.SSC (2.times.standard salt solution--0.3 M NaCl, 30 mM Na
citrate, pH 7.0) were chosen. For each gene, a pair of primers was
generated with the help of Primer3 software (Rosen and Skaletsky
(1998) Primer3. Code available at
http://www-genome.wi.mit.edu/genome software/other/primer3.html.).
The program parameters were chosen in such a way that the melting
temperature of the amplicon should be close to 80.degree. C. but
not more than 88.degree. C. or less than 75.degree. C., the length
of the amplicon was to be generally around 450 bp (with a few
outlyers between 300 and 700 bp), with primer annealing temperature
about 60.degree. C., and average length of primers 23 bp. Sequences
of all amplicons have been carefully verified using proprietary
software (BLASTN, FASTA), to avoid homology with repetitive
elements and other related sequences, and also to distinguish
between genes from the same family. A full list of all selected
genes is represented in Table 1.
[0057] DNA, RNA and mRNA Isolation
[0058] Total RNA and DNA were isolated from approximately 10.sup.8
HeLa cell cultures and human peripheral lymphocytes isolated from
fresh blood aliquots using, Trizol reagent (Gibco BRL, Burlington,
ON). DNA and RNA concentrations and quality were determined by
spectrophotometric and gel electrophoresis analysis in 0.8 or 2%
agarose gels, respectively. Poly(A).sup.+RNA was isolated from 150
.mu.g of total RNA using the Oligotex mRNA kit (Qiagen,
Mississauga, ON), according to the manufacturer's instructions.
[0059] Amplification and Purification of Probes
[0060] 10 .mu.g of total RNA was reverse-transcribed in 40 .mu.l
reaction, using 200 U of MMLV (Gibco BRL, Burlington, ON) according
to the manufacturer's instructions. Two PCR reactions for each pair
of primers were conducted in a total volume of 100 .mu.l, in a
GeneAmp PCR system 9700 (PE Applied Biosystems, Norwalk, Conn.).
Each 50 .mu.l reaction (10 mM Tris-HCl, pH8.6, 50 mM KCl, 0.1%
Triton X-100, 1.5 mM MgCl.sub.2, 0.5 mM of each dNTP, 20 pM of each
primer, 1.25 U of Taq DNA polymerase (Amersham Pharmacia Biotech,
Baie d'Urfe, QC) and 10 .mu.l of RT reaction or 100 ng of genomic
DNA) was thermal-cycled as follows: first cycle at 94.degree. C.
for 5 min, 35 cycles at 94.degree. C. for 45 sec, at 60.degree. C.
for 1 min and at 72.degree. C. for 30 sec, the last cycle at
72.degree. C. for 7 min. Probes that could not be amplified in
RT-PCR were extracted from genomic DNA, with the condition that the
primers were selected in the 3' region of a gene. Size and yield of
PCR products were determined by gel electrophoresis in 2% agarose.
Then PCR products were purified from solution or agarose gel bands,
following preparative agarose gel electrophoresis (if by-products
were determined), using GFX columns (Amersham Pharmacia Biotech,
Baie d'Urfe, QC). After purification, concentrations of all probes
were estimated by agarose gel electrophoresis, and adjusted to
approximately 100 ng/.mu.l.
[0061] Robotic Arraying
[0062] Purified PCR products in 2.times.standard salt solution
(SSC) were arrayed in triplicates from 384-well plates, utilizing a
GeneMachines.TM. OmniGrid microarrayer (Genome Instrumentation
Services, San Carlos, Calif.) equipped with ChipMaker2 tips
(Telechem International, San Jose, Calif.). The spacing between
dots was 400 .mu.m. The positions of genes in this array are
indicated in Table 4.
[0063] Microarrays were printed on Hybond-N or Hybond-N+ nylon
membranes (Amersham Pharmacia Biotech, Baie d'Urfe. QC), attached
to standard glass slides with tape. Before and after each 10 slides
with membranes, regular slides were inserted to inspect printing
quality. After arraying, membranes were UV irradiated at 50 mJ (GS
Gene linker, Bio-Rad, Hercules, Calif.) to immobilize the DNA; then
fragments of membranes containing arrays (approximately 1.times.1.5
cm) were cut off, denaturated in boiling water for 5 min, rinsed in
0.1% SDS for 5 min, and used for prehybridization. After the UV
irradiation step, membranes can be stored attached to glass
slides.
[0064] Preparation of DIG-Labeled cDNA for Hybridization
[0065] An initial mix of gene-specific primers (GSP) was produced.
For this purpose, 1 .mu.l of each primer that was used in RT-PCR
reactions to prepare probes was mixed in a total volume of 250
.mu.l. Digoxigenin (DIG)-labeled targets were produced in RT
reaction as follows: 1 .mu.l of GSP, 4 .mu.g of total RNA, and
RNAse-free water in total volume of 14 .mu.l were heated at
65.degree. C. for 15 min to denature the RNA, and then kept at room
temperature for 5 min for primer annealing. Alternatively, 2 .mu.g
of mRNA and 400 ng of oligo(dT).sub.12-18 primers were used. The
reaction mix, containing 8 .mu.l of 5.times.first strand buffer
supplied by the enzyme's manufacturer, 2 .mu.l of 10 mM mix of
dATP, dCTP and dGTP (final concentration 500 .mu.M each), 4 .mu.l
of 0.1 M DDT, 0.7 .mu.l RNAguard, 31 U/.mu.l (Amersham Pharmacia
Biotech, Baie d'Urfe, QC), 10 .mu.l of a 2 mM mix of 19:1
dTTP:DIG-11-dUTP (Roche, Laval, QC) and 2 .mu.l (200 U/.mu.l) of
Moloney murine leukemia virus reverse transcriptase (MMLV RT)
(Gibco BRL, Burlington, ON), was added. Reaction was carried out at
37.degree. C. for 1 h, followed by enzyme degradation at 94.degree.
C. for 5 min in GeneAmp 9700. Alternatively, Omniscript reverse
transcriptase (Qiagen, Mississauga, ON) was used according to the
manufacturer's instructions. Labeling reactions were purified on
GFX columns; this step eliminates all labeled products shorter than
100 bp, as well as unincorporated nucleotides, primers and
protein.
[0066] After purification, efficacy of labeling was estimated as
follows: 1 .mu.l of 1:100, 1:1000, 1:10000 and 1:100000 dilutions
were spotted on Hybond-N membrane, together with dilutions of
control DIG-labeled DNA at known concentrations (10-0.01 pg/.mu.l)
as standardization for our assays (Roche, Laval, QC); after
immobilization with UV, the membrane was incubated with alkaline
phosphatase (AP)-conjugated antibody to DIG (Anti-DIG-AP), rinsed,
and stained with chemiluminescent substrate, Disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1-
.sup.3,7]decan})-4-yl) phenyl phosphate--CSPD (Roche, Laval, QC),
according to the manufacturer's instructions.
[0067] Hybridization and Processing
[0068] For hybridization and pre-hybridization, DIG Easy Hyb buffer
(Roche, Laval, QC), or formamide buffer containing 50% deionized
formamide, 5.times.SSC, 2% blocking solution (Roche, Laval, QC),
0.1% N-lauroylsarcosine, 0.02% SDS, 100 .mu.g/ml denaturated salmon
DNA, were used. Membranes were pre-hybridized at 42.degree. C. for
2 h in a hybridization oven (Autoblot, Bellco, Vineland, N.J.).
Hybridization was performed at 42.degree. C. overnight in 1 ml or
less of hybridization solution, in 5-ml Falcon tubes. The
concentration of labeled probes in the hybridization mix
constituted 10 ng/ml. Before hybridization the probes were
denaturated at 65.degree. C. for 10 min in hybridization
solution.
[0069] Afterwards, hybridization membranes were rinsed (unless
mentioned specially) twice with 1.times.SSC, 0.1% SDS for 15 min at
room temperature, and then with prewarmed 0.1.times.SSC, 0.1% SDS
for 15 ml at 68.degree. C. Alternatively, membranes were rinsed in
more stringent conditions, i.e. twice in 2.times.SSC, 0.1% SDS at
68.degree. C. for 30 min, and twice in 0.1.times.SSC, 0.1% SDS at
68.degree. C. for 30 min. After equilibration for 5 min in rinsing
buffer (0.3% Tween 20 in maleic buffer (0.1 M maleic acid, 0.15 M
NaCl, pH 7.5)), membranes were blocked for 1.5 h in 1% blocking
solution under slight agitation, and then treated for 30 min in 10
ml of alkaline phosphatase-conjugated sheep anti-digoxigenin
antibody (Roche, Laval, QC), diluted 1:1000 for colorimetric
staining, or 1:10000 for chemiluminescent detection. Following
antibody incubation, membranes were rinsed three times for 15 min
in rinsing buffer, equilibrated for 2 min in detection buffer (0.1
M Tris-HCl, 0.15 M NaCl, pH 9.5), and stained with 175 .mu.g/ml
5-Bromo-4-chloro-3-indolyl-phosphate, toluidine salt (BCIP), and
330 .mu.g/ml Nitro blue tetrazolium chloride (NBT) in detection
buffer. Alternatively, 1:100 dilution of CSPD was applied, and
chemiluminescence was detected according to the manufacturer's
recommendations (Roche, Laval, QC) using BioMax MR Kodak film.
[0070] Scanning and Evaluation of Arrays
[0071] Arrays were scanned on an Olympus microscope equipped with a
Multiscan-4 System (Applied Scientific Instrumentation, Eugene,
Oreg.) and a color CCD Sony 950 camera. Data acquisition and
montage of different fields of view into one file were accomplished
with the help of the Northern Eclipse Imaging System (EMPIX
Imaging, Missisauga, ON). Quantitative measurements of intensity of
enzymatic reaction at each dot, background subtraction,
normalization to housekeeping genes, and comparison of paired
hybridizations were all performed with an in-house software
program.
[0072] Results
[0073] Selection of Probes and Primers
[0074] After careful evaluation of different data bases, 61 genes
were selected for arraying, including 9 housekeeping genes. This
set of genes contains 38 E-box binding genes, together with the Myc
(c-, N-, L1 and L2) family, 5 c-Myc regulating factors (ZFP161,
nm23-H2S, MBP-1, RBMS 1 and RBMS2), 5 c-Myc interacting genes (YY1,
TFII-1, PAM, MM-1 and alpha-tubulin), and 4 c-Myc target genes
(prothymosin alpha, MRDB, ODC1, and cdc25A). Positive controls
include 9 housekeeping genes with different levels of expression
(UBC, beta-actin, GADPH, HPRT1, phospholipase 2, HLA-C, PRS9,
aldolase C, and RPL13A), and also HeLa genomic DNA. Lambda DNA and
2.times.SSC (2.times.standard salt solution), which was used as
solvent for all probes, were selected as negative controls.
[0075] Printers for all genes were selected with the help of
Primer3 software, provided that they corresponded to the same
conditions for PCR reaction, and produced products of similar
melting temperature. Most products were produced from HeLa or
lymphocyte cDNA. In case PCR amplification failed from cDNA,
primers were selected in 3' region of these genes, and amplicons
were produced from HeLa genomic DNA. The average annealing
temperature of primers was 60.1.+-.0.9.degree. C., which allowed
all PCR reactions to be in the 96-well format. Sizes and melting
temperatures of products, and annealing temperatures of primers,
are represented in Table 4. The average size of PCR products for
arraying, and their melting temperature, were 441.+-.58 bp and
80.+-.3.degree. C., respectively. Selecting these parameters
allowed hybridization and post-hybridization rinsing in stringent
conditions, decreasing drastically the possibility of
cross-hybridization and background level.
[0076] Scrupulous selection of primers could be used to distinguish
in some cases between very close members of gene families (for
example, USF1 and 2, ID2, 3 and 4, members of the Myc family, and
so on), or between two different transcripts of c-Myc. As is well
known, there are several different transcription forms of c-Myc,
transcribed from different promoters, with varying regulation
properties (Bodescot and Brison Gene 174, 115-120 (1996)).
Selecting primers in the 1.sup.st exon and the 2.sup.nd-3.sup.rd
exons allowed discrimination between full-size and truncated forms
of c-Myc.
[0077] Conditions Influencing Hybridization
[0078] Several parameters which probably influence the results of
hybridization with cDNA microarrays printed on nylon membranes were
carefully tested. First of all, gene profiling results were
examined using either mRNA or total HeLa RNA. Surprisingly, the
whole pattern of expression was very similar, with the exception of
a few genes (UBC, RPL-13A, MBP-1) the signals from mRNA were
several times higher; the most prominent difference was found in
UBC, where it approached 5-fold. Alternatively, signals for HPRT1
and phospholipase A2 were higher with total RNA. In conditions
where quantity of mRNA is a limiting factor, total RNA can be used
instead, without significant differences in results of expression
profiling.
[0079] Comparison of two reverse transcription enzymes, Moloney
murine leukemia virus (MMLV) (Gibco BRL, Burlington, ON) and
OmniScript (Qiagen, Mississauga, ON), used for production of
digoxigenin-labeled targets for hybridization, did not reveal any
difference in expression profile when gene-specific primers were
used; but signal intensity was stronger after labeling with MMLV,
especially after a day of staining (Table 5). When oligo(dT)
primers were used with mRNA, some significant differences in
expression levels of several genes were detected. Labeling with
OmniScript produced 2-3 times more intense signals for RP-S9,
RP-L13A, enolasel, N-Myc and MAD4.
[0080] To decide which buffer is better for hybridization with
microarrays, we compared EasyHyb (Roche, Laval, QC) and
formamide-based buffers. The expression profile of HeLa mRNA was
found to be independent of buffer composition, but signals were
higher after hybridization in formamide buffer (Table 2), and
addition of 2% blocking reagent further reduced background in
comparison with EasyHyb, thereby facilitating subsequent scanning
and image evaluation.
[0081] No substantial differences were found in expression profile
of HeLa mRNA when rinsing conditions of different stringency were
used (see Materials and Methods). More stringent rinsing evenly
lowered all signals, and produced signals with sharper borders,
rendering them easier to scan and evaluate. Standard rinsing
conditions are probably already stringent enough in hybridizations
with cDNA microarrays and gene-specific primers; therefore standard
rinsing is preferred, because it is not so time-consuming.
[0082] Comparison of positively charged (Hybond-N+) with neutral
(Hybond-N) nylon membranes revealed no differences in sensitivity.
Aside from this consideration, the neutral (Hybond-N) nylon
membrane is preferable due to its stronger texture for printing
support. This strength was not found in the positively charged
Hybond-N+ membrane, which was found to retain visible printing
footprints, causing complications in image analysis and increased
background.
[0083] As may be seen from Table 5, increasing the staining time
from overnight to 1 day usually increased the overall strength of
signals by only 10%. Longer staining time increased the background
level of the reaction, which compromised the possible advantage of
higher sensitivity. Variations in hybridization conditions can
increase overall signal intensity by 30-40%. However, the positive
effects are not additive, and the maximum difference in total
intensity of microarrays approaches only 50%. The following
conditions for hybridization of DIG-labeled targets with the cDNA
microarray are optimal: printing probes on neutral nylon membrane,
reverse transcription reaction with total RNA, genie-specific
primers and MMLV reverse transcriptase, hybridization in formamide
buffer, and standard rinsing conditions. These conditions were
implemented in the experiments described in the following
paragraphs.
[0084] Specificity, Sensitivity and Reproducibility of
Hybridization
[0085] To evaluate the specificity of cDNA microarray
hybridization, 5 genes (MRDB, ODC, TFII-1, cdc25A and c-Myc),
covering the entire range of length (368-711 bp) of arrayed
products, were labeled in multiplex PCR reaction and hybridized
with cDNA arrays. As expected, only 5 samples on the array were
positive, as well as the HeLa genomic DNA as control since it will
hybridize with the locus where HeLa genomic DNA was spotted at the
highest concentration at the position 51, and negative show little
or no detection at the positions 1a and 1b where spotted HeLa
genomic DNA is of low quantity. In all, these experiments
demonstrate no signs of cross-hybridization (FIG. 3).
[0086] To estimate the sensitivity and derive a calibration curve
for cDNA microarray hybridization, different concentrations of this
5-gene PCR mix (10, 4, 1, 0.4, 0.1 and 0.04 ng/ml) were hybridized
with arrays. The results of this experiment are presented in FIG.
4. Linear dependence in semi-logarithmic coordinates, with an
obvious plateau in the region of 4-10 ng/ml, was observed for all
genes, with the same slope of 45.+-.2. The lower limit of detection
varies slightly for different probes in the array, and corresponds
to 40-100 pg/ml per individual gene. These results are close to the
detection limit of the digoxigenin system (10-30 pg/ml), according
to the manufacturer (Roche, Laval, QC). This level of sensitivity
allows detection of mRNAs of intermediate abundance, each
representing more than 0.04% of total cell mRNA. Taking into
account this detection level, it is estimated that for
hybridization with a microarray containing about 70 genes of
intermediate abundance, 7 ng of labeled probe produced from
gene-specific primers should suffice. For the next hybridizations a
concentration of labeled probes of 10 ng/ml was selected. The yield
of standard reverse transcription labeling reaction with genie
specific primers is about 20-40 ng; therefore, one labeling
reaction yields enough product for 2-4 independent hybridization
reactions. In contrast to unstable radioactive probes. DIG-labeled
probes can be stored and reused several times. Reusing
hybridization mixes 2-3 times, after storing at -20.degree. C. for
several months, gave results quite concordant with the original
ones.
[0087] The arrays were scanned at a resolution of 3600 dpi, and
results were compared with results of microscope scanning. In
general, variability between replicated dots was higher in the case
of the scanner, and linearity may be influenced by the scanner's
software. The scanner can be used for initial evaluation of
hybridization results, especially when chemilumenescence detection
is implemented.
[0088] Expression Profiling of Hela Cells in Comparison with Human
Lymphocytes
[0089] Expression profiles of E-box genes were determined in
replicating HeLa cells and normal human lymphocytes. In
lymphocytes, the most prominent alteration consisted of more than
2-fold up-regulation of E-box-related genes TCF4, MAD4 and Aldolase
C. Alternatively, down-regulation of c-Myc-regulating genes MBP1
and Nm23-H2S, and small down-regulation of c-Myc and up-regulation
of N-Myc, were registered in lymphocytes in comparison with HeLa
cells. Expression of some c-Myc interacting and target genes was
down-(MM-1, ODC1) or up-regulated (PAM, MrDb) in lymphocytes. Also,
small up-(MITF, ID2) and down-(TFEB) regulation was detected in
expression of several E-box-binding genes in lymphocytes, in
comparison with HeLa cells.
SUMMARY
[0090] cDNA and oligonucleotide microarrays are becoming an
increasingly powerful technique for investigating gene expression
patterns. In spite of the fast progress in this field, some
limitations of the technique persist. One of the major obstacles is
the requirement for a large amount of mRNA. Another problem with
existing microarray systems is data mining; while information on
expression of tens of thousands genes is absolutely vital to
estimate the functions of new genes, in some instances a researcher
is interested in the expression profile of only a subset of genes,
in many physiological conditions. The significant differences ill
expression of 3-6 genes out of 61 are already much more manageable
than can be detected from ordinary microarrays with massive numbers
of genes, in the hundreds or thousands. For example, in SAGA
analysis of 45,000 genes, it was found that about only 1% are
differentially expressed in normal and cancerous human cells. A
similar estimation resulted from analysis of expression profiles in
young and old mice; expressions of only 1.8% of about 6,000 genes
are changed more than 2-fold.
[0091] Printing microarrays on nylon filters, and using digoxigenin
to label the cDNA with gene-specific primers, permits use of as
little as 4 .mu.g of total RNA per hybridization. This is the same
sensitivity that can be attained with radioactivity in the Clontech
protocol, and it is much more sensitive than ordinary microarrays,
which need several .mu.g of mRNA. In addition, DIG-labeled probes
of high labeling sensitivity can be stored for a long time, and
reused several times, in contrast to fluorescently or radioactively
labeled ones.
[0092] Proprietary selection of genes for inclusion in a
microarray, and using digoxigenin for labeling, also helps avoid
another disadvantage of radioactive labeling: genes in the E-box
microarray are all in the same category of abundance (intermediate
or low abundant). Excluding highly abundant genes eliminates the
problem of merging of strong signals. Merged signals in some
circumstances substantially complicate the process of scanning, and
create unreliable results during the data acquisition step.
* * * * *
References