WHAT IS CUSTOMARRAY™?
CustomArray™ is a unique system for the in situ synthesis of oligonucleotide microarrays made to your specifications. You design the CustomArrays using web-based free software; we ship them to you 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 CUSTOMARRAY DIFFERENT FROM SPOTTED ARRAYS?
CustomArray is an in situ synthesis technology. Oligonucleotide probes are synthesized directly on the chip. This means there are no up front costs of buying hundreds of different oligonucleotides, and making changes to chip designs and layouts is easy.
HOW IS CUSTOMARRAY DIFFERENT FROM OTHER IN SITU SYNTHESIS TECHNOLOGIES?
CustomArray chips use electrochemical detritylation to control DNA synthesis. Electrochemical detritylation is a much more efficient method to synthesize oligonucleotides than light-based synthesis processes. This means the oligonucleotides are of the highest quality and the sensitivity of the microarray is maximized. 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 costs.
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.
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.
WHAT METHOD IS USED FOR QUALITY CONTROL OF CUSTOMARRAY CHIPS?
Each and every synthesized CustomArray chip is quality tested by hybridization 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 to ensure the high quality of chips shipped to our customers. The arrays that have passed the inspection are stripped of the hybridized 9-mers, washed, and dried for shipment.
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. 2), and low coefficients of variation among replicate probes within the same array (on average from 6 to 10%).
Figure 2. 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 CHIPS?
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. 3).
Figure 3. 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.
WHAT SPECIAL EQUIPMENT DO I NEED TO USE CUSTOMARRAY?
You will require a microarray scanner to image the chips after processing. CustomArray is compatible with a number of imaging systems that have been developed for spotted microarrays. We recommend a slide-based, focusable scanner with a minimum of 5 um resolution. Axon, Perkin Elmer, and BioRad all have models compatible with our chips (Axon GenePix 4000B, GenePix 4200A, GenePix 4200AL; Perkin Elmer ScanArray® 4000 and 5000, ScanArray® Lite, ScanArray® Express). Contact a technical support representative at CombiMatrix if you have questions regarding the compatibility of your imaging system.
You may also need a rotisserie hybridization oven to rotate arrays during hybridization. CombiMatrix produces a variety of array holders for many common oven types used in the labs. All other materials for processing should be readily available in a basic molecular biology lab.
WHAT MATERIALS ARE SHIPPED TOGETHER WITH CUSTOMARRAY 12K CHIPS?
Each CustomArray chip comes with the following items:
Hybridization chamber
Gasket (O-ring)
Two clamps
LifterSlip™ coverslip for imaging (Lifterslip™ is a trademark of Erie Scientific Corporation)
Imaging solution for array scanning.
All these material are intended for single use.
WHAT SORT OF MATERIAL DO I NEED TO WORK WITH CUSTOMARRAY?
For each chip, you will need to prepare 5-10 micrograms of biotinylated or fluorescently labeled nucleic acid (genomic DNA, cDNA, cRNA) using your method of choice. Our technical support can provide some guidelines depending on your type of array application.
HOW MANY PROBES/FEATURES CAN I PUT ON A CUSTOMARRAY CHIP?
CustomArray 12K allows you to choose 12,000 distinct probes per array. The arrays also have a set of 544 factory-built control probes for quality control purposes.
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.
HOW LONG ARE THE OLIGONUCLEOTIDE PROBES ON A CUSTOMARRAY?
Oligonucleotide probe length depends on application. For gene expression studies we recommend to use 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 probes is set at 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 HAVE FACTORY-BUILT CONTROLS?
Yes, all CustomArray chips carry a set of factory-built controls including two types of probes: negative controls (NC), and quality controls (QC). In the factory layout, they are positioned as blocks in different areas around the chip. A factory layout scheme with labeled pre-built controls is shown in Figure 4. Several probe blocks are left as ‘no-synthesis’ controls for QC purposes.
Both NC and QC probes could be used in your experiments as negative controls for background calculation, providing that they do not cross-hybridize with your material. The NC probes labeled "plant," "bacteria," and "phage" are derived from Arabidopsis thaliana, Agrobacterium tumifaciens, and phage lambda, respectively. The exact sequences of these probes are shown in Table 1. You could BLAST these control sequences against your genome of interest to estimate cross-hybridization. If you work with plant and bacterial genomes, you may need to design your own additional set of negative controls instead of using the pre-built ones.
Figure 4. The factory layout of pre-built control probes (shown as red squares).
Table 1: CustomArray 12K chip: factory-built control probes.
CAN I REPLACE THE FACTORY-BUILT CONTROLS WITH MY OWN CONTROL PROBE SET?
No. These probes are essential for array quality control that is done individually for each synthesized chip after manufacturing.
WHAT SPIKING CONTROLS CAN I USE TO ESTIMATE CUSTOM ARRAY PERFORMANCE?
The factory-built controls from our standard factory layout can be used as spiking probes, providing that they do not cross-hybridize with your material. The sequences of these probes are shown in Table 1. You can order oligonucleotides that are reverse-complimentary to the selected control probes, and get them end-labeled (biotin or fluorescent dye, should be the same labeling as in the planned experiments). Two or three of these oligonucleotides would be sufficient as spiking controls to add to the hybridization mixture.
Phage lambda spiking controls could be prepared and used with the NC probes named ‘NC-SP-phage’ that are included in the standard factory layout. Preparation of lambda spiking controls from commercial phage lambda DNA includes several steps: PCR amplification of lambda DNA fragments, cloning into a vector containing the T7 RNA-polymerase promoter, sequencing of the cloned fragments, in vitro transcription, and quality control of the resultant RNA.
You can also use commercially available Stratagene spiking control mRNA samples made from 7 Arabidopsis thaliana genes. Typically two or three spiking controls should be sufficient. You need to select them and include the corresponding probes from the following list into your array design files.
Please note that these controls would cross-hybridize if you work with plant material.
WHAT NEGATIVE CONTROLS CAN I USE?
Negative control probes are usually used to calculate background for subtraction. The area surrounding spots and no synthesis spots cannot be used for background calculation because they give an over-estimation of background. You can use factory-built probes (both negative, NC, and quality controls, QC) as negative controls, providing that they do not cross-hybridize with your material. The NC probes labeled "plant," "bacteria," and "phage" are derived from Arabidopsis thaliana, Agrobacterium tumifaciens, and phage lambda, respectively. If you work with any of these organisms, you may need to provide your own set of negative controls for probe design.
Plotting the distribution of NC spot intensities can show how well particular NC probes perform in the context of you array. For background subtraction, we suggest to use only NC probes with low intensities (lowest 5-30%). If your arrays do not have an appropriate set of negative controls with low signal intensities, it might be better to omit the background subtraction step from data analysis.
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.
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.
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?
Recommendations for hybridization buffers and temperatures are outlined in our ‘Hybridization Protocol for CustomArray™12K Chips & Preparation for Scanning', and they are designed for gene expression analysis. Other applications may require some optimization of these parameters. Please contact our technical support specialists for further suggestions.
HOW MUCH NUCLEIC ACID DO I NEED FOR THE HYBRIZATION?
You need 5 microgram biotinylated or fluorochrome-labeled nucleic acid per array per color channel (10 microgram for the dual-color experiments).
DO I NEED TO ROTATE ARRAYS DURING HYBRIDIZATION?
Yes. Usage of a rotisserie oven or a rotating incubator is highly recommended to ensure mixing during hybridization. You can introduce a small air bubble into the hybridization chamber to improve the mixing process during rotation. CustomArray chips can be attached to standard rotisseries using various models of holders manufactured by CombiMatrix. If you plan to buy a new rotisserie hybridization oven, we recommend Fisher Scientific Isotemp Hybridization Incubators which can be outfitted with a large CombiMatrix rotisserie holding 32 arrays.
CAN I STRIP THE HYBRIDIZED TARGET NUCLEIC ACIDS AND RE-USE CUSTOMARRAY CHIPS?
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 targers 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.
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™12K Chips & Preparation for Scanning’ 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)
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)
WHAT AM I SEEING ON MY SCANNED IMAGE?
Restrict the scan area to the array part (semiconductor chip) to minimize scanning time. You will see a regular pattern of round circles of varying intensity which correspond to the discrete features on the array. 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 cannot be used for background calculations (please see below for recommendations on background calculation).
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 MY SCANNED IMAGE?
CustomArray™ 12K has 224 rows and 56 columns of spots.
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?
Background calculation for CustomArray chips should be based on a set of negative controls that do not hybridize with your material. Spots with no synthesis, or the area outside of spots should not be used, as these will give an over-estimation of background. You can provide your own set of negative controls, or use the CustomArray set of factory-built control probes. Our set includes probes named negative controls (NC), and quality controls (QC), and both may be used, providing that they do not cross-hybridize with your material. The NC probes labeled "plant," "bacteria," and "phage" are derived from Arabidopsis thaliana, Agrobacterium tumifaciens, and phage lambda, respectively. If you work with plant and bacteria, you may see significant cross-hybridization with our factory-built controls.
Plotting the distribution of NC and QC spot intensities can show how well particular probes perform in the context of your material. For background calculation, we suggest to use only control probes with low intensities (lowest 5-30%), and then to calculate their median signal intensity that can be used for subtraction. All 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 you plan to compare (not to create artificial ratios).
If your arrays do not have a special set of negative controls with low signal intensities, you may use the lowest 5% of signals, or omit the background subtraction step from data analysis.
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.
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.
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.
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 a short period of time (maximum a couple of days), providing that they are kept wet, preferably with the hybridization chambers filled with 2x PBS (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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