WHAT
IS CustomArray 4x2K?
CustomArray 4X2K is based on the same semiconductor platform as
CustomArray 12K, it is a microarray that is divided into 4 sectors,
each of which can contain up to 2,240 different oligonucleotide probes
(spots). Sectors may be hybridized individually with different targets
using the provided sectored hybridization cap. Oligonucleotide probes
for CustomArray 4X2K are custom designed to user specifications by
using web-based free software. Microarrays are then generated based on
CombiMatrix technology (hyperlink) of in situ synthesis and shipped to
customers within 30 business days.
HOW DOES CUSTOMARRAY
WORK?
The custom oligonucleotide microarray is synthesized on a semiconductor
using an electrochemical synthesis process. Each oligonucleotide probe
(spot) is synthesized on a platinum electrode that is independently
controlled by the synthesizer’s computer. Synthesis is based on
established phosphoramidite chemistry and occurs at thousands of sites
simultaneously according to a computer algorithm that activates only
specified electrodes. Since physical photolithographic masks,
mechanical micromirrors, or inkjet technologies are not involved in the
process, all probes can be easily changed without extra time or cost.
The oligonucleotides are synthesized in a
proprietary porous reaction layer that coats the surface of the
semiconductor, and acts to covalently attach the oligonucleotide to the
surface of the chip. The resultant CustomArray chip is shown in Fig.1.
Figure 1. A general scheme of CustomArray 12K chips.
HOW
IS CUSTOMARRARY 4X2K DIFFERENT FROM CustomArray 12K?
CustomArray 4X2K is a sectored version of CustomArray 12K, and the
basic technology is the same: in situ synthesis of oligonucleotide
probes directly on the microarray. Sectors may be hybridized
individually with different targets using the provided sectored
hybridization cap.
DOES
THE SECTORED CAP CHANGE CustomArray PERFORMANCE IN COMPARISON WITH THE
STANDARD CAP?
CustomArray 4X2K is a sectored version of CustomArray 12K, and it is
shipped with the sectored hybridization cap, instead of the standard
one provided for the 12K microarray. CombiMatrix hybridized 4x2K
CustomArray twice with the same material using the two different types
of hybridization caps. As shown in Fig. 2, in both cases the resultant
data were very consistent.
Fig.
2. CustomArray 4X2K were hybridized with the same labeled cRNA targets
using two different types of hybridization caps: the standard one for
12K microarrays, and the sectored one for 4x2K microarrays. Pair-wise
correlation coefficients (r2) were calculated between corresponding
sectors using raw data (no normalization, no background correction).
Scatter plots show comparisons of log-transformed data.
WHAT CAN I USE CUSTOMARRAY 4X2K
FOR?
CustomArray 4X2K can be used for the same applications as CustomArray
12K, including gene expression studies, comparative genome
hybridization (CGH), SNP analysis and detection, re-sequencing, and
chromatin immunoprecipitation (ChIP) on chip. CustomArray 4X2Kis
divided into 4 sectors, each of which can contain up to 2,240 different
oligonucleotide probes (spots). Sectors may be hybridized individually
with different targets using the provided sectored hybridization cap.
However, all 4 sectors must have the same probe content and layout
design. Thus, one 4x2K microarray can be used to analyze 4 different
samples simultaneously using the same oligonucleotide probes.
HOW LONG DOES IT TAKE TO MAKE MY
OWN CustomArray?
After you have finalized your array design, we typically ship the
finished arrays within 30 days from receipt of your order.
CAN CustomArray BE
RE-USED AFTER THE FIRST HYBRIDIZATION?
A single CustomArray microarray can be stripped three times using the
CombiMatrix CustomArray Stripping Kit. The CustomArray Stripping
protocol is based on chemical denaturation of DNA:DNA and DNA:RNA
hybrids between oligonucleotide probes on microarrays and corresponding
labeled targets. As a result of this denaturation, labeled targets are
removed from oligonucleotide probes and washed off microarrays. The
resultant stripped CustomArray microarrays can be used in the same way
as newly synthesized ones. In total, the CombiMatrix Stripping Kit
would enable you to use a single CustomArray microarray four times.
CustomArray Quality and Performance
WHAT
METHOD IS USED FOR QUALITY CONTROL OF CustomArray 4X2K CHIPS?
CombiMatrix uses the same quality testing for both 12K and 4X2K. It
means that each and every synthesized microarray and sector is
hybridized with Cy5-labeled random 9-mer oligonucleotide targets. This
hybridization is used to visualize the synthesis at every electrode as
well as irregular spot morphology. All hybridization data are first
analyzed automatically using rigorous statistical criteria, and then
inspected visually by an independent reviewer for the final 'pass' or
'fail' decision. All 4 sectors should pass the inspection for a 4x2K
microarray to be shipped to our customers.
HOW
SENSITIVE ARE CUSTOMARRAYS?
We hybridized CustomArrays with a complex labeled target mixture
containing four different spiking control RNA’s added at concentrations
of 0.375, 0.75, 1.5, and 3 pM. The resultant data showed reproducible,
above-background signals for the lowest spike concentration (0.375 pM),
linearity at the applied concentration range (Fig. 3), and low
coefficients of variation among replicate probes within the same array
(on average from 6 to 10%).
Figure
3. Hybridization of spiking controls to 2 CustomArray chips in the
context of a complex labeled target mixture. Four different spiking
control RNAs were added at concentrations of 0.375, 0.75, 1.5, and 3
pM. The average standard deviation across the set of replicate probes
is shown by the error bars.
HOW REPRODUCIBLE ARE CustomArray MICROARRAYS?
We hybridized 20 identical CustomArray chips with the same RNA target
sample prepared from the Universal Human Reference RNA (Stratagene) and
labeled with Cy3 and Cy5 according to the dual-color scheme. Our raw
data (no background correction) showed the following average
correlation coefficients among different pairs of 20 chips: 0.97 for
the Cy3 color channel, and 0.98 for the Cy5 color channel. In total,
97% of all pair-wise correlation coefficients were over 0.95.
Each probe was synthesized in
triplicate, so we estimated coefficients of variation for replicate
probes on the same array. In total, less than 3% of all genes had
coefficients of variation exceeding 20%.
Our arrays were hybridized
with the same material labeled in Cy3 and Cy5, so we calculated a false
positive discovery rate based on 2-fold change in these 20
same-versus-same comparisons. Among 247,920 ratio measurements, we
observed 551 ratios equal or exceeding 2-fold, thus, only 0.22% of
false positives.
Such high between- and
within-array reproducibility and low false positive rate have been
observed with raw data prior to background correction. Background
correction and simple ‘global’ normalization bring pair-wise
correlation coefficients between Cy3 and Cy5 color channels on the same
chip to average 0.992 (minimum 0.985) resulting in highly consistent
data (Fig. 4).
Figure 4. Scatter plots
of 15 replicate arrays hybridized with the same material (Universal
Human Reference RNA) labeled with Cy3 and Cy5. For each array, X-axis
is Log2(Cy3 Intensity). Y-axis is Log2(Cy5
Intensity). Raw data has been normalized and subjected to background
correction.
HOW
DOES PERFORMANCE OF CustomArray 4X2K COMPARE WITH CustomArray 12K?
CustomArray 4X2K is based on the same semiconductor platform CustomArray12K, and has very similar performance in terms of
chip-to-chip reproducibility (average correlation coefficients of
0.97).
WHAT
IS DATA REPRODUCIBILITY BETWEEN CustomArray 4X2K AND 12K?
The main difference between CustomArray 12K and 4x2K is usage of two
different types of hybridization caps: a standard one for 12K
microarrays, and a sectored one for 4x2K microarrays. Fig. 2 shows that
usage of the sectored cap doesn’t change CustomArray performance.
Pair-wise correlation coefficients (r2 )
between the data obtained with two types of hybridization caps are
shown in Fig. 2, and the average r2 is 0.97
(calculated for raw data without normalization and background
correction).
WHAT
IS REPRODUCIBILITY BETWEEN INDIVIDUAL SECTORS OF THE SAME CustomArray
4X2K?
CombiMatrix hybridized each 4 sectors of the CustomArray 4X2K with the
same RNA target sample, and analyzed raw data without normalization and
background correction. As shown in figure 5, all four sectors of each
microarray had very similar mean signal intensities, with coefficients
of variation (CV) in the range from 4 to 11% (Fig. 5). As shown in Fig.
6, the average pair-wise correlation coefficient (r2)
among different sectors on the same microarray was 0.97. When the data
were log-transformed, the correlation coefficients r2
between individual sectors further increased to 0.98-0.99. Scatter
plots of log-transformed raw signal intensities comparing sectors
within the same array are shown in Fig. 7. Practically all data points
showed very small changes between individual sectors (please note very
tight distribution of data points). CombiMatrix calculated a false
positive discovery rate based on 2-fold change between sectors
hybridized with the same material. On average, 1 to 3 data points
showed ratio of changes over 2-fold, and this was less than 0.15% of
all probes in an individual sector.
Fig.5. Mean
signal intensities and coefficients of variation (CV) calculated for
each individual sector within the CustomArray 4X2K. Calculation was
done for raw data (no normalization, no background correction).
Fig.6. Pair-wise
correlation coefficients (r2) calculated between
individual sectors within 4 of CustomArray 4X2K. The mean r2=0.97.
Calculation was done for raw data (no normalization, no background
correction).
Fig.7. Scatter plots of
log-transformed raw signal intensities comparing individual sectors
within 2 of CustomArray 4X2K (first two rows are from microarray #1,
and the second two rows from microarray #2). The mean r2=0.99.
Calculation was done without normalization and background correction.
Design
WHAT
MATERIALS ARE SHIPPED TOGETHER WITH CustomArray 4X2K CHIPS?
Each CustomArray chip comes with the following items:
Sectored hybridization cap (chamber)
LifterSlip™ coverslip for imaging (Lifterslip™
is a trademark of Erie Scientific Corporation)
Imaging solution for array scanning.
All these materials are intended for single use,
though the hybridization cap could be washed and re-used for several
hybridizations.
DO
I NEED TO BUY ADDITIONAL MATERIALS FROM COMBIMATRIX TO USE WITH CustomArray 4X2K?
A 4X2K hybridization clamp, which is a proprietary metal device that
holds CustomArray 4X2K microarray and hybridization cap together
during hybridization and washing will need to be purchased. Please note
that this hybridization clamp is used only for 4x2K and not for 12K
microarrays. The hybridization clamp is intended for multiple use.
Purchasing an array holder for your laboratory’s
rotisserie oven is also an option. This array holder attaches the
CustomArray 4X2Ks assembled in the hybridization clamps. CombiMatrix
produces a variety of array holders for most commercially available
oven types. Contact a technical support representative at CombiMatrix
for advice on what type of holder will work for specific hybridization
ovens. Please note that the rotisserie holders for CustomArray 4X2Ks
are different from the 12K ones, and they are not interchangeable. The
appropriate holder is necessary because the rotation axis is different
for the 4X2K and 12K microarrays.
WHAT
SPECIAL EQUIPMENT DO I NEED TO USE CustomArray 4X2K?
A 4X2K hybridization clamp, which is a proprietary metal device that
holds the 4X2K microarray and hybridization cap together during
hybridization and washing will need to be purchased. A rotisserie
hybridization oven to rotate arrays during hybridization may also be
purchased. CombiMatrix produces a variety of array holders for many
commercially available oven types. Please note that the rotisserie
holders for CustomArray 4X2K and 12K are different,and they are not
interchangeable.
A microarray scanner is required to image the
microarrays after processing. CustomArray is compatible with a number
of imaging systems that have been developed for spotted microarrays.
CombiMatrix recommends a slide-based, focusable scanner with a minimum
of 5 um resolution. Axon, Perkin Elmer, BioRad, and VIDAR Systems all have models
compatible with our microarrays (Axon GenePix 4000B, GenePix 4200A,
GenePix 4200AL; Perkin Elmer ScanArray4000, ScanArray5000, ScanArray
Lite, ScanArray Express; VIDAR Systems Revolution® 4550). Contact a technical support representative at
CombiMatrix for questions regarding the compatibility of imaging
systems.
All other materials for processing should be
readily available in a molecular biology lab.
WHAT SORT OF MATERIAL DO I
NEED TO WORK WITH CustomArray 4X2K?
For each sector of each microarray, prepare 1-2 micrograms of
biotinylated or fluorescently labeled nucleic acid (genomic DNA, cDNA,
cRNA) using the method of choice. CombiMatrix technical support can
provide some guidelines depending on the type of array application. The
email address for technical support is support-at- combimatrix.com.
HOW MANY PROBES/FEATURES
CAN I PUT ON A CustomArray 4X2K?
CombiMatrix requests that all 4 sectors of a given microarray have the
same probe design and layout. CustomArray 4x2K allows the user to
choose 2,240 distinct probes per one array sector. Please note that
control probes for normalization and background correction may need to
be provided by the user. CustomArray 4x2K is initially synthesized on
the CustomArray platform with a standard set of factory-built control
probes for quality control purposes. However, these controls are
located as 3 blocks positioned between 2,240-spot sectors, and will be
completely covered by the gasket of the sectored hybridization cap.
Thus, the factory-built controls will become inaccessible for your
hybridization.
CAN
I CHANGE PROBE DESIGNS AND LAYOUTS AMONG SECTORS OF THE SAME CustomArray 4X2K?
No, at present CombiMatrix requests that all 4 sectors of a given
microarray have the same probe design and layout. Probes and designs
can be changed from microarray to microarray, but not within the same
sectored microarray.
ARE THERE ANY TOOLS FOR
PICKING LISTS OF GENES?
Genome sequencing data is available at The National Center for
Biotechnology Information (National Institute of Health) http://www.ncbi.nlm.nih.gov.
Tools that classify genes by functional categories are available at The
Cancer Genome Anatomy Project http://cgap.nci.nih.gov. This
system can be used to browse the Gene Ontology database and produce
lists of human and mouse genes. Importing lists of genes from
pre-existing data sets, or converting Unigene identifiers to accession
number formats can also be accomplished in batch using conversion tools
such as Matchminer http://discover.nci.nih.gov/matchminer.
WHAT
STEPS ARE INCLUDED IN THE COMBIMATRIX PROBE DESIGN ALGORITHM?
You can provide a file with GenBank accession numbers or nucleotide
sequences that correspond to genes of interest. Our probe design
software applies a set of proprietary algorithms that is based on the
generally accepted probe selection rules, and trained from experimental
data obtained with CustomArray chips. This software will perform
several subsequent iterations to select probes that are unique within
the defined gene set, are located in the specified part of genes, have
Tm and length within the specified range, do not form stable secondary
structure, and do not carry repeat sequences.
In addition, you can provide an ‘excluded targets’
file containing GenBank accession numbers or nucleotide sequences that
should not cross-hybridize with your probes. Typically, these sequences
correspond to highly abundant contaminants such as ribosomal RNA. It is
also possible to use this function to distinguish genes from a
homologous gene family, by inputting common parts of sequences as
excluded targets.
During the probe design process, the probe
specificity can be enhanced by screening the probe for uniqueness
within the known transcriptome. This additional screen is achieved
using a non-redundant database, Unigene, which contains a single entry
for each known gene of a particular genome. This screen is done
automatically after you specify the type of background organism from a
pull down menu (you may select up to three background organisms for one
run). In some cases, no relevant Unigene screen can be performed since
these databases are only available for a subset of organisms.
CAN
I CUSTOMIZE THE PROBE DESIGN ALGORITHM FOR ARRAY APPLICATIONS OTHER
THAN GENE EXPRESSION?
Our CustomArray chips can be used for a variety of different
applications that may require changes in the probe selection
algorithms. Our Design-on-Demand® service can guide you through this
process and help to customize the probe design algorithm to your need.
Please see information on Design-on-Demand
or email
for more information.
CAN I PUT MY OWN PROBES ON THE
ARRAY?
Yes. In addition to allowing the software to design probes for you, you
can also populate the chips with your own probes. For optimum
performance, you need to keep the length and Tm of the customer-defined
probes within the same range as for the rest of probes on the chip.
IS
THERE ANY DIFFERENCE BETWEEN PROBE DESIGN ALGORITHMS FOR CustomArray
4X2K IN COMPARISON TO THE 12K?
CombiMatrix uses the same algorithm to design probes for both types of
CustomArrays. Please contact our Design-on-Demand service for help in
customizing the probe design process to fit your needs. The
Design-on-Demand Team can be reached at design-at-combimatrix.com.
HOW LONG ARE THE
OLIGONUCLEOTIDE PROBES ON A CUSTOMARRAY?
Oligonucleotide probe length depends on the desired application. For
gene expression studies CombiMatrix recommends using 30 to 40-mer
probes. SNP analysis and re-sequencing may require short, 18 to 25-mer
probes to maximize mismatch sensitivity. The upper size limit for CustomArray 4X2K probes is 50-mer.
CAN I MIX GENOMES, SUCH AS
COMBINING PROBES TO VIRUS AND HOST GENES, ON ONE CHIP DESIGN?
Yes. Probes to multiple genomes can be used as long as the Tm of the
probes is within the same range (we recommend within five degrees for
optimal performance). You can specify up to three background
organisms/genomes for an automatic screening for probe uniqueness.
WHAT SORT OF CONTROLS DO I
NEED?
You can use spiking controls, negative controls, and positive controls,
such as housekeeping genes. Labeled spiking controls can be used to
evaluate array performance (between- and within-array variation), they
may be added to the labeled target samples prior to the hybridization
and should be in the 1-100 pM range. Negative controls should be
included for background calculation purposes. Housekeeping genes may be
used for normalization among arrays.
DOES CustomArray 4X2 HAVE FACTORY-BUILT CONTROLS?
Each 4x2K is synthesized on the CustomArray platform with a standard
set of factory-built control probes for quality control purposes.
However, these controls are located as 3 blocks positioned between
2,240-spot sectors, and will be completely covered by the gasket of the
sectored hybridization cap. Thus, the factory-built controls will
become inaccessible for hybridization. Users may need to provide
control probes for normalization and background correction.
WHAT
SPIKING CONTROLS CAN I USE TO ESTIMATE CustomArray PERFORMANCE?
Commercially available Stratagene spiking control mRNA samples
(http://www.stratagene.com/products/) made from 7 Arabidopsis
thaliana genes can be used. Typically 2 or 3 spiking
controls should be sufficient, select them and include the
corresponding probes from the following list into the array design
files.
Please note that these controls would
cross-hybridize if you work with plant material.
WHAT
NEGATIVE CONTROLS CAN I USE FOR 4X2K CustomArray?
Negative control probes are usually used to calculate background for
subtraction. The area surrounding spots and no synthesis spots can not
be used for background calculation because they give an over-estimation
of background. In contrast to the12K, the 4x2K does not make available
the factory-built probes because they are covered by the gasket of the
sectored hybridization cap and are inaccessible for hybridization.
Thus, users may need to provide their own set of negative controls.
Alternatively, the background can be calculated from the lowest 1-5% of
signal intensities on the 4X2K. This value may then be used for
background subtraction. Please note that this approach may not work, if
you optimize the array content in several iterations to select for
probes with high hybridization signals. In such situation, the
background subtraction step may be omitted, or a set of negative
controls added.
WHAT
POSITIVE CONTROLS CAN I USE?
You can provide a list of housekeeping genes for positive control probe
design. It should include genes with minimum variation in the
expression levels under the applied experimental conditions. Thus, it
is impossible to provide general recommendations applicable to all
systems. However, a list of potential candidates for human arrays can
be obtained here.
You need to select a relatively large set of
housekeeping genes to make the housekeeping gene approach less
sensitive to outlier data, because even such 'constantly' expressed
genes as actin still tend to have some variation in expression levels.
In addition, you need to keep this set constant for all arrays that you
plan to compare to each other. We recommend using at least 50 different
genes with sufficient numbers of replicates (at least three per gene).
Ideally, your set of housekeeping genes should contain both high and
low expressed ones to cover the full range of signal intensities
(except for the extremely high, close to saturation, and extremely low,
close to background levels).
CAN
I USE RIBOSOMAL RNA SEQUENCES AS POSITIVE CONTROLS?
We do not recommend it for gene expression studies in eukaryotic
organisms, because the standard mRNA amplification methods (based on
oligo(dT) primers) should dramatically reduce the proportion of rRNA in
the sample. This reduction may be non-uniform for different samples,
different amplification reactions, and different inputs of total RNA.
Variable results of this selection against rRNA make it a poor control
for array data normalization.
Hybridization
HOW
MUCH STARTING MATERIAL (RNA OR DNA) DO I NEED FOR AN ASSAY WITH
CUSTOMARRAY CHIPS?
CustomArray chips have been shown to work with a wide variety of
labeled target nucleic acids produced using different commercial kits.
So the amount of initial material that you need to start with depends
on the applied technique for target amplification and labeling. For
example, T7 polymerase-based RNA amplification kits (e.g. from Arcturus
and Ambion) result in very high degrees of amplification (especially
when used in double rounds). Thus you may start with as little as 100
ng of total RNA and still get enough material to hybridize with
CustomArray chips.
WHAT TYPE OF NUCLEIC ACIDS
WORKS BEST FOR HYBRIDIZATION?
Both RNA and DNA labeled target samples work in hybridization assays.
However, the hybridization conditions should be different, please see
our Hybridization Protocol (in .pdf
format).
CAN
I USE BOTH SINGLE-COLOR AND DUAL-COLOR HYBRIDIZATION APPROACHES? HOW DO
I LABEL NUCLEIC ACID TARGETS FOR HYBRIDIZATION WITH CUSTOMARRAY CHIPS?
Yes, both approaches work for CustomArray chips. For the single-color
approach we recommend to label nucleic acid targets by biotin
incorporation, to hybridize arrays with the biotinylated targets, and
then to perform post-hybridization labeling using
streptavidin-conjugated fluorescent dye (e.g. Fluorolink Cy5-labeled
streptavidin from Amersham, Catalog # PA45001). For the dual-color
approach, two target nucleic acid samples may be directly labeled with
two different fluorescent dyes (e.g. Cy3 and Cy5) during amplification
reactions using commercially available kits (e.g. Ambion kits for cRNA
and cDNA labeling). For gene expression applications with eukaryotic
material we recommend to use the Ambion MessageAmp™ II aRNA
Amplification Kit (Catalog #1751).
Direct incorporation of fluorescent dye labels
into nucleic acids results in short shelf life of the labeled targets
which have to be utilized within one month from labeling. Only
biotinylated targets can be stored as long as unmodified nucleic acids.
The storage problem can be alleviated if you amplify and store nucleic
acid targets without labeling, and then apply a quick direct labeling
technology using Label IT® reagents from Mirus (http://www.mirusbio.com)
to obtain samples for array hybridization. This technology is based on
binding of a label to the guanine residues in DNA and RNA in a fast
non-enzymatic reaction. You can select a Label IT® Kit with a
fluorescent dye (e.g. Cy3 and Cy5) or biotin, and get your target
samples ready for hybridization in approximately 2 hours.
WHAT IS THE OPTIMUM TEMPERATURE
FOR HYBRIDIZATION OF CustomArray 4X2K?
Recommendations for hybridization buffers and temperatures are outlined
in Hybridization Protocol for CustomArray 4X2K
& Preparation for Scanning, and they are designed for
gene expression analysis. Other applications may require some
optimization of these parameters. Different hybridization buffers can
be used for different sectors of the 4X2K microarray.
DO
I NEED TO HYBRIDIZE ALL FOUR SECTORS OF A 4x2K MICROARRAY AT THE SAME
TIME?
Yes, it is best to hybridize all four sectors of a CustomArray 4X2K at
the same time. If you don’t use all four sectors, the un-used
microarrays will become wet during scanning, because you need to scan
the whole surface under a single LifterSlip™. Subsequent drying may
cause damage to unused sectors of the array. Thus, after scanning the
only remaining storage option is to keep all four sectors of a
CustomArray 4X2K wet until the next hybridization. You can store
CustomArray 4X2K in 1xPBS or the imaging solution at 4°C (to prevent
microbial growth).
HOW MUCH NUCLEIC ACID DO I NEED
FOR THE HYBRIZATION?
CombiMatrix recommends usage of 1 to 2 microgram of labeled targets per
one array sector. If two labeled samples are hybridized to the same
array (the dual-color hybridization scheme), use 1 to 2 microgram of
each sample (totally 2-4 microgram).
DO
I NEED TO ROTATE CustomArray 4X2KS DURING HYBRIDIZATION?
Yes, usage of a rotisserie oven or a rotating incubator is highly
recommended to ensure mixing during hybridization. When working with
4x2K, air bubbles don’t need to be introduced into the hybridization
chambers, just fill the chambers completely and seal them with tape.
CombiMatrix produces a variety of array holders
for many common oven types used in the labs. If buying a new rotisserie
hybridization oven, CombiMatrix recommends Fisher Scientific Isotemp
Hybridization Incubators, which can be outfitted with CombiMatrix array
holders. Please note that the rotisserie holders for CustomArray 4X2Ks
and 12K’s are different and are not interchangeable.
CAN
I STRIP THE HYBRIDIZED TARGET NUCLEIC ACIDS AND RE-USE CustomArray
MICROARRAYS?
A single CustomArray microarray can be stripped three times using the
CombiMatrix CustomArray Stripping Kit. The CustomArray Stripping
protocol is based on chemical denaturation of DNA:DNA and DNA:RNA
hybrids between oligonucleotide probes on microarrays and corresponding
labeled targets. As a result of this denaturation, labeled targets are
removed from oligonucleotide probes and washed off microarrays. The
resultant stripped CustomArray microarrays can be used in the same way
as newly synthesized ones. In total, the CombiMatrix Stripping Kit
would enable you to use a single CustomArray microarray four times.
Imaging and Data Analysis
HOW DO I IMAGE MY ARRAY?
The CustomArray chip should be imaged “wet” to obtain the best
signal-to-noise ratio. A special imaging solution is provided with each
array shipment. Each array is also provided with a special LifterSlip™
coverslip that allows the array to be imaged under buffer. The
LifterSlip™ is used to protect the array from damage resulting from
direct contact with the glass, and to prevent excess liquid from
damaging the scanner. Please see our Hybridization
Protocol for CustomArray 4X2K for recommendations on how to
prepare arrays for scanning. It is critical to image the array in the
correct orientation with the chip facing the optics. It is not possible
to image the array through the slide.
CAN I USE MY OWN COVERSLIP
FOR IMAGING?
No. The LifterSlip™ must be used, or some damage may occur to the
array. The two raised edges, with white coating on the sides, prevent
the LifterSlip from directly touching the surface of the array. The
LifterSlip provides a 50 micron high cavity above the chip surface.
WHAT
SCANNERS DO I USE?
CombiMatrix recommends the following scanners:
Axon GenePix™ 4000B, 4200A, and 4200AL
AppliedPrecision: arrayWoRx®e Biochip Reader
PerkinElmer: ScanArray® 4000, ScanArray® 5000,
ScanArray® Lite, ScanArray® Express (please contact our technical
support about applicability of ScanArray Gx ®, ProScanArray®,
ProScanArray® HT)
VIDAR Systems Revolution® 4550
Other scanner models may also be used providing
that they have two essential features:
scan resolution of 5 micron
adjustable focus (manual, or the auto-focusing
feature)
CAN
I IMAGE ONLY A PART OF CustomArray 4X2K?
No, image the whole microarray using one large LifterSlip provided.
WHAT AM I SEEING ON THE 4X2K
SCANNED IMAGE?
Restrict the scan area to the array part (semiconductor chip) to
minimize scanning time. A regular pattern of round spots of varying
intensity which correspond to the discrete features on the array can be
seen. Each spot is located within a dark square or rectangle bounded by
elements of the semiconductor chip. These elements show up as a visible
grid that does not normally interfere with data extraction. In any
case, the area outside of spots can not be used for background
calculations (please see below for recommendations on background
calculation).
A typical image of CustomArray 4X2K is shown in
Fig. 8, four distinct sectors with hybridized spots (2,240 each)
separated by three dark blocks (1,232 spots each) with no hybridization
signals can be seen. The dark blocks contain factory-built controls
that have been covered by the sectored hybridization cap and thus are
unavailable for hybridization.
Fig.8. A typical image
of CustomArray 4X2K, all four sectors are hybridized with the same
labeled target RNA sample. The image is obtained by scanning with Axon
4000B scanner, and data extraction is done using CombiMatrix Microarray
Imager.
HOW
LARGE IS ONE SPOT ON CUSTOMARRAY?
Each spot is 44 micron in diameter, and the distance between centers of
two neighbor spots is 75 micron. Thus, one square of the semiconductor
grid that appears on CustomArray images is also 75 micron (Fig. 1).
HOW
MANY ROWS AND COLUMNS OF SPOTS AM I SEEING ON THE 4X2K SCANNED IMAGE?
Each sector of CustomArray 4X2K is composed of 40 rows and 56 columns
of hybridized spots. Each dark block separating sectors is composed of
22 rows and 56 columns of factory-built controls inaccessible for
hybridization.
CAN
I USE COMBIMATRIX SOFTWARE FOR DATA EXTRACTION?
Yes, we provide a free software application, Microarray Imager, for
data extraction; it can open most image formats. The detailed guide on
how to use it is located on our applications
website.
CAN
I USE THIRD-PARTY SOFTWARE FOR DATA EXTRACTION?
Yes, you can use data extraction software provided with your scanner.
In some cases, the appearance of a semiconductor grid around the spots
may cause problems with autotemplating. Our technical support team may
have solutions for some of these problems, or you can try our
Microarray Imager for data extraction.
HOW
DO I OBTAIN THE CHIP DESIGN FILE SHOWING POSITIONS OF PROBES ON MY
ARRAY?
A chip design file is a file in XML format that contains information
about your probes and their location on your CustomArrays. You can
access this file though our applications website by
registering and selecting "My Designs." Our data extraction software,
Microarray Imager, will directly accept the XML chip design file, so no
conversion is required. CombiMatrix also provides a tool for converting the chip design files
into GAL files. GenePix and some other programs for array
data extraction will accept the GAL format. CombiMatrix can assist
customers in converting to other file formats as well.
HOW DOES THE 12K CHIP DESIGN
FILE WORK WITH MICROARRAY IMAGER?
CombiMatrix stores probe location information in XML format in a file
called a chip design file. Microarray Imager understands this file and
can open most image formats, so no conversion of the input files is
required to extract data from images of our microarrays.
HOW DO I USE OTHER MEANS OF
IMAGING BESIDES THE MICROARRAY IMAGER?
CombiMatrix provides a tool for converting its chip design files into
GAL files. GenePix and some other programs for extracting data from
microarrays will accept this format. CombiMatrix can assist customers
in converting to other file formats as well.
HOW DO I EXTRACT DATA FROM MY
ARRAY?
The chip design file and the image of the hybridized chip from the
microarray scanner need to be opened in Microarray Imager (available on
the applications
website). It is critical to open the correct design file with
the correct chip image; the design file will have the annotations
associated for that specific chip. Microarray Imager will associate the
raw intensities for each spot on the chip with the appropriate
annotations once the data is extracted. Follow the instructions for
Microarray Imager available on this website.
HOW
DO I PERFORM ARRAY DATA NORMALIZATION?
Between-array normalization is performed to compensate for technical
variability. In most cases, a simple ‘global’ normalization technique
would be sufficient for CustomArrays. For each array, the median signal
intensity is calculated based on all probes. Then scale factors are
calculated for all arrays in the analyzed set to bring their median
signal intensities to the same level.
Several other normalization techniques may also be
used, such as the ones based on (1) a set of ‘housekeeping’ genes and
(2) probes with low intensity (the lowest 1-5% of signal intensities).
The housekeeping gene approach works if you are confident that the
selected genes are more or less constantly expressed in your material,
and if the gene set is large enough to compensate for outliers (at
least 100 of genes/probes). If the initial data normalization is done
based on a limited gene set, they need to be excluded from further data
analysis because the resultant normalized data for these genes could be
no longer considered statistically independent.
Background correction may be done after
normalization is completed.
HOW DO I CALCULATE AND SUBTRACT ARRAY BACKGROUND FOR CUSTOMARRAY 4X2K?
Negative control probes are usually used to calculate background for
subtraction. The area surrounding spots and no synthesis spots can not
be used for background calculation because they give an over-estimation
of background. In contrast to the 12K, users can not use the
factory-built probes of the 4X2K because they are covered by attachment
of the sectored hybridization cap and are inaccessible for
hybridization. Thus, users may need to provide their own set of
negative controls. Alternatively, background from the lowest 1-5% of
signal intensities on the array can be calculated. Please note that
this approach may not work, if you optimize the array content in
several iterations to select for probes with high hybridization
signals. In such situation, background subtraction step may be omitted,
or add a set of negative controls.
CombiMatrix recommends calculating a background
value and standard deviation of background for each array, and then to
subtract background from individual data points. All resultant negative
values are then converted into a common default value (it can be set as
2x or 3x standard deviations of background). This default should be the
same for all arrays that are compared (not to create artificial
ratios). All data points that are below 2x or 3x standard deviations of
background may also be converted into the same default value.
WHAT SOFTWARE SHOULD I USE TO
INTERPRET CUSTOMARRAY RESULTS?
Data from Microarray Imager can be exported into a variety of third
party analysis software packages. Note that some formatting may be
required to upload data into third party software.
On Chip Assays
WHAT CAN I USE
CUSTOMARRAY FOR?
CustomArray can be used to order whole microarrays just like ordering
oligos! Initially, our platform has been tailored for gene expression
studies. At present, it is widely used for other applications,
including comparative genome hybridization (CGH), SNP analysis and
detection, re-sequencing, and chromatin immunoprecipitation (ChIP) on
chip. The web-based
system for designing chips can create optimized probes based
on a set of customer-defined sequences that are submitted as GenBank
accession numbers or raw sequence data. It can also accept
customer-specified oligonucleotide probe sequences. Our Design-on-Demand service
can help to change the probe design algorithm to tailor probes for your
particular application.
CAN I MAKE
DOUBLE-STRAND PROBES ON THE CUSTOMARRAY?
Yes. By incorporating a common linker at the 3' ends of all probes
(proximal to the array surface), one can anneal a primer that is
complementary to the linker and extend it with a DNA polymerase (such
as Klenow fragment of E. coli DNA polymerase I)
in buffer containing the dNTP mix.
CAN I MAKE ENZYMATIC ASSAYS
ON THE CHIP?
Yes, you can fill the hybridization chamber with an appropriate buffer
and perform most enzymatic reactions on the chip. You can phosphorylate
the 5’ ends of probes with T4 polynucleotide kinase, extend primers on
the probes with polymerases, ligate primers to probes with T4 DNA
ligase or E. coli DNA ligase, and perform restriction endonuclease
digestions, etc. This ability to perform enzymatic reactions on our
chips is the basis for development of new assays, such as Hyb &
Seq used for SNP analysis and re-sequencing. Please see Design-on-Demand
for more information.
Software
WHAT IF INTERNET EXPLORER
DOES NOT LAUNCH THE COMBIMATRIX SOFTWARE?
Java Web Start needs to be installed. If Java Web Start is not
installed, Internet Explorer will usually show a message that it cannot
open a file of type JNLP.
If Java Web Start has been installed and Internet
Explorer still will not launch CombiMatrix Software, it may be
necessary to clear the Internet Explorer temporary files.
WHAT OPERATING SYSTEMS DO I NEED TO
RUN COMBIMATRIX SOFTWARE?
CombiMatrix recommends Microsoft Windows XP or Microsoft Windows 2000.
Storage
WHAT IS THE SHELF LIFE OF A
CUSTOMARRAY?
Four months.
HOW SHOULD I STORE CUSTOMARRAY
CHIPS?
In a dark, dust free, dry environment at room temperature, or in a
dessicator.
CAN
I STORE HYBRIDIZED ARRAYS?
We do not recommend storage of hybridized arrays because it may cause a
decrease in signal and data quality. However, the hybridized arrays may
be stored for short periods of time, providing that they are kept wet,
in 1xPBS or the imaging solution, well protected from light (wrapped in
foil), and at low temperature (4-8°C in refrigerator).
If you have questions that have not been answered
here, please email:
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