Arrayit primary nucleotide sequence of specific long oligonucleotides present in the H25K Human Genome Microarray.
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Arrayit primary nucleotide sequence of specific long oligonucleotides present in the H25K Human Genome Microarray. Customers can obtain raw sequence information for one or more of the 26,304 oligonucleotides that comprise the H25K product. Arrayit revolutionary one spot-one gene™ design utilizes the most advanced bioinformatics and the complete human genome sequence to obtain fully optimized H25K oligonucleotides that are highly gene specific. This convenient program allows users to obtain some or all of the H25K design simply by purchasing the oligonucleotide sequence information. Ideal for users studying specific human gene subsets. Customers should specify the unique 5-digit TCHA numerical identifier for each sequence (e.g. 07265) at the time of ordering. TCHA identifiers for each oligonucleotide sequence can be found in the H25K Human Genome Annotation file available on-line.
Reagents - DNA Microarrays - H25K Human Genome Microarrays, Oligonucleotide Sequences, and Complete Human Genome Expression Profiling Services
Arrayit has pioneered H25K, the world’s first and only human genome microarray derived from the complete human genome sequence. H25K contains a complete set of 25,509 fully annotated human genes representing every gene in the human genome. Our next generation H25K microarray offers the highest sensitivity and specificity of any product on the market, owing to the use of long-mer oligonucleotides, proprietary attachment and surface chemistry and a unique one gene-one spot design. H25K allows customers to decipher orphan disease pathways, benchmark drugs, delineate the molecular basis of known diseases and gain deeper access to the human transcriptome. H25K is fully compatible with the entire Arrayit line of instruments and consumables installed in more than 3,800 laboratories worldwide. The H25K chip will yield molecular markers for the most challenging studies in molecular medicine. Arrayit also offers complete human genome expression services and Biomarker Discovery and Validation Services for customers wishing to obtain novel H25K disease and drug markers on a services basis.
The use of ArrayIt® brand products will improve the precision, speed and affordability of your microarray research in genomics, proteomics, biomedicine, pharmaceuticals and diagnostics. This handbook contains all the information required to take full advantage of Arrayit's H25K Human Genome Microarrays.
Arrayit uses the highest quality control (QC) and quality assurance (QA) measures to ensure the quality of our ArrayIt® brand H25K Human Genome Microarrays. The finest science and engineering was used to develop this product. The use of advanced bioinformatics, ultra-pure oligonucleotides, superior surface chemistry and the world’s most advanced microarray cleanroom manufacturing facilities guarantees that H25K outperforms the highest industry standards.
Arrayit H25K Human Genome Microarrays represents the most advanced human genome microarray in the industry. H25K was designed using the complete, fully annotated human genome sequence using sophisticated one spot-one gene™ informatics, and has the highest gene content on the market (see Figure 1). H25K is manufactured in state-of-the-art class 1 cleanrooms using our patented contact printing technology (U.S. 6,101,946), with the strictest QC and QA procedures. H25K offers quality and performance that cannot be obtained from any other microarray or next generation sequencing approach.
Users will appreciate the following features of H25K
Designed from the complete, fully annotated human genome sequence
Highest gene content of any human genome chip on the market
Annotated across GenBank, UniGene, GoldenPath, RefSeq and AceView
Contains a total of 26,304 long oligonucleotide elements
Novel one spot-one gene™ design streamlines data analysis
Explore the function of 25,509 verified human genes
Devoid of pseudogenes, and redundant and chimeric sequences
A total of 795 control sequences add precision and reliability
Contains widely used Ambion and Stratagene controls
Multiple uses including karyotyping, comparative genomic hybridization (CGH), expression analysis, and karyotyping
Compare human, primate and rodent genomes
Detect >300,000 human mRNAs in a single hybridization reaction
Purchase one or all of the 26,304 oligonucleotide sequence set
Map chromatin domains genome-wide
Manufactured using Arrayit’s patented printing technology (U.S. 6,101,946)
Oligonucleotides made “off-line” exceed 99% purity levels
Content verified using mass spectrometry to ensure integrity
Printed on atomically smooth glass substrates for improved precision
Proprietary series 3 Arrayit glass offers world’s lowest intrinsic fluorescence
Compatible with an installed base of >10,000 slide scanners
Use H25K to validate Affymetrix, Agilent, ABI, GE and other vendors
Printed elements occupy a convenient 18 x 54 mm area
50 µl reaction volume accelerates kinetics and minimizes sample consumption
Chip contains 48 sub-grids (24 x 24) of 125 µm printed elements
Format compatible with all standard data quantification and mining packages
Compatible with dozens of DNA and RNA amplification and labeling schemes
Utilize fluorescent, colorimetric and chemilluminscent detection
Produced in our automated manufacturing facilities
Microarrays arrive processed and ready to use
Stable for 6 months from the date of purchase
Explore the entire human genome in a single experiment
H25K was used by Arrayit scientists to decipher Parkinson’s Disease
Figure 1. Arrayit H25K Human Genome Microarrays contains the highest Genbank and AceView human gene content of any product on the market.
Each H25K Human Genome Microarray kit contains the following components:
1 x H25K Human Genome Microarray
1 x Human Discover Chip or equivalent
1 x H25K Lifter cover slip (22 x 60 mm)
1 x Discover Chip cover slip (22 x 22 mm)
1 x H25K Human Genome Annotation (downloadable using the link below)
Figure 2. H25K Human Genome Microarray kit manufactured and packaged in class 1 cleanrooms and heat-sealed in anti-static packaging to ensure optimal performance for 6 months from the date of purchase. Kit contents include an H25K Human Genome Microarray, a Human Discover Chip, and cover slips. Complete H25K gene annotation information is available electronically using the download links.
The ArrayIt® H25K Human Genome Microarray contains 26,304 features or “spots” representing 25,509 fully annotated human genes, as well as 795 control sequences from mouse, plant and other sources. H25K oligonucleotides contain SENSE strand information, and therefore nucleic acid probe mixtures reacted with H25K must contain ANTI-SENSE molecules to hybridize to the SENSE oligonucleotides present on the chip. The long oligonucleotide sequences present in each feature are annotated across the AceView, Entrez, RefSeq, UniGene, KnownID, and GenBank databases. Each oligonucleotide also includes a unique MicroarrayID, which identifies the sequence on H25K and allows customers to purchase the sequence by specifying the MicroarrayID in the catalog number (see Ordering Information). Gene descriptions are based on the best descriptions available from GenBank and AceView. A complete description of the annotation information is provided in Table 1.
Table 1. Provided are descriptions of the H25K annotation information.
Microarray specific identifier for H25K. Use the last 5 digits to order H25K oligonucleotide sequences (see Ordering Information)
The H25K content is printed as 48 sub-grids of 24 x 24 spots spaced at 185 µm centers. The printed H25K microarray occupies an 18 x 54 mm area on the glass substrate, positioned evenly positioned from left to right and top to bottom between the top of the substrate and the barcode. The 12.5 x 19 mm barcode includes the product name, website, barcode, and unique 6 digit tracking number (e.g. 100012) as shown in Figure 3. The barcode information is included to allow unambiguous H25K sample tracking at the customer site. Barcode information is also used at the Arrayit manufacturing centers for quality control and quality assurance purposes. One major advantage of H25K compared to competing human genome microarrays is that our patented contact printing method and glass substrates allow visual inspection of every printed H25K chip, guaranteeing the presence of the printed elements. This contrasts with in situ synthesis approaches, which produce microarrays with “invisible” synthesized elements that are not amenable to direct inspection. The H25K inspection process on a microarray-by-microarray basis provides the customer with a level of QA and QC that is not possible with competing products.
Figure 3. H25K Human Genome Microarray with barcode and lifter slip. The H25K microarray, lifter slip and 75 µl probe mixture should be pre-heated to 42°C for 5 min before applying the probe to ensure low and uniform background. The top edge of the barcode label can be used to gently lower the lifter slip onto the probe 75 µl droplet. A properly positioned lifter slip containing the 75 µl probe mixture is shown here.
Short Protocol (Steps 1-7)
1. Prepare a labeled probe from a biological sample.
2. (Optional) Test the labeled probe mixture on a Human Discover Chip.
3. React the probe sample with H25K to allow molecular binding.
4. Wash H25K to remove unbound probe molecules.
5. Detect H25K signals by scanning or imaging.
6. Quantify the H25K signals.
7. Perform data analysis.
Complete Protocol (Steps 1-7)
1. Prepare a labeled probe from a biological sample. H25K contains 26,304 long oligonucleotides representing every gene currently known in the human genome. These gene-specific hybridization targets can be used to explore the genome, transcriptome and proteome in a variety of applications. Probes can be prepared from human DNA, mRNA and protein samples, or from homologous species such as primates and rodents. Sample mixtures can be complex and of native origin or from synthetic recombinant sources, such as in cases where H25K is being used to map “contigs” or artificial chromosomes. H25K can be used to query a single probe mixture, or multiple probes prepared using multiple fluorescent labels in multi-color DNA and RNA hybridization. For gene expression applications, each oligonucleotide designed to be complimentary to the 3’ end of a gene and is optimized to hybridize to as many transcripts as possible in cases where genes express multiple mRNAs. H25K can detect as many as 300,000 human mRNAs in a single reaction. There are three fundamental principles that should be used for H25K sample preparation: (1) Sample integrity must be high. DNA, mRNA and protein molecules must be full length and free of contaminating enzymes that can produce nucleic acid and protein degradation. (2) Probe labeling must be efficient. Probes should be checked prior to reactions with H25K to ensure that the probe mixtures are highly fluorescent, or contain the expected epitopes such as amino-allyl or biotin. The most common cause of poor performance with H25K is poor sample/probe preparation. (3) Probe mixtures must be pure. Prior to reactions with H25K, probe mixtures should be purified to remove non-incorporated dyes and other contaminants that can increase background noise. This step is well worth the small investment in time and money, as it will greatly increase performance. Direct and indirect fluorescent and non-fluorescent labeling techniques have been described extensively in the literature and a host of commercial labeling kits are available from many different vendors. Arrayit offers an extensive line of purification, amplification and labeling kits for DNA, mRNA and protein labeling applications. For gene expression applications, Arrayit’s Micro-Total RNA Extraction Kit, MiniAmp mRNA Amplification Kit, Indirect Amino Allyl Fluorescent Labeling Kit, and Universal Reference mRNA are highly recommended for maximum signal strength.
2. (Optional) Test the labeled probe on a Human Discover Chip. Each H25K Human Genome Microarray Kit includes a Human Discover Chip, which is a test microarray containing long oligonucleotides complementary to 380 human genes. To open the H25K Kit, cut the silver anti-static bag between the heat seal and the zip-lock approximately 1.0 cm below the top end of the package using a scissors. This will allow the silver package to be re-sealed if only a portion of the H25K Microarrays is used. The silver package contains a white box with H25K and the Discover Chip, and a thin plastic cassette that houses the cover slips. To open the white H25K box, lift the top gently upward while holding the bottom firmly in the other hand. Once the box is opened, remove the white cleanroom cloth by lifting it gently upward. Avoid dragging the cleanroom cloth across the glass substrates as this may cause fraying of the cloth. The Human Discover Chip contains a 9 x 9 mm microarray located on the upper third of the glass substrate. A portion of a probe mixture destined for H25K can be tested on the Human Discover Chip in advance of using the more expensive H25K product. Probes mixtures prepared from DNA, RNA and protein (5-10 µl) can be applied to the Human Discover Chip using the 22 x 22 mm included in the H25K Kit. Make sure to open the thin plastic cassette carefully as the cover slips are fragile and can be broken if mishandled. Use H25K steps 3-7 below as a guide for the Human Discover Chip test experiment. The Discover Chip annotation can be downloaded from the Human Discover Chip website. Step 2 is optional, and users with a high confidence in their technique and probe quality may omit this step and proceed directly to using H25K.
3. React the probe sample with H25K to allow molecular binding. The 26,304 printed elements of H25K are located in an 18 x 54 mm area on the barcode side of the glass substrate. For additional help with side orientation, the 1.4 mm cropped corner of the glass substrate (“chamfer”) should be located in the upper right corner of the glass substrate when the barcode is facing upward. The Figure 3 photograph shows the side of the glass substrate that contains the H25K microarray. Do not attempt to react probe mixtures with the backside of H25K, as negative results will be obtained. Gloves should be worn at all time while handling H25K to avoid the transfer of dust, debris, hand oils, and other contaminants that can interfere with H25K use. ArrayIt® Microarray Cleanroom Gloves provide superior hand protection for microarray applications. The recommended probe sample volume for H25K is 75 µl, which will form a 60 µm sample layer on the microarray when using a 22 x 60 mm lifter cover slip. The recommended hybridization buffer is HybIt® 2 Hybridization Solution. Arrayit provides an affordable and widely used line of Hybridization Cassettes for H25K probe sample reactions, and Hybridization Cassettes work very well for low to medium throughput applications. Users may also employ the Arrayit TrayMix™ S4 Automated Hybridization Stations to allow automated reactions and washing of H25K (Fig. 4). Probe mixtures can be generated from cloned or cellular DNA, RNA or protein, and dozens of fluorescent and non-fluorescent labeling schemes have been described in the scientific literature. For gene expression applications with H25K, 2.0 µg of amino-allyl labeled cDNA per color (4.0 µg total of green and red probe for two-color experiments) is recommended to produce strong hybridization signals. Probe mixtures should be spun at 18,000 x g for 1 min to pellet debris prior to use. The H25K substrate slide, hybridization cassette or chamber, lifter slip and probe mixture should be pre-heated to 42°C for 5 min prior to hybridization to ensure low and uniform background. Pipette the 75 µl probe mixture directly onto the center of the microarray and quickly place the lifter slip onto the probe droplet, allowing the probe mixture to sheet evenly across the surface. Lay the bottom end of the lifter slip on the top edge of the barcode to allow air bubbles to escape, then nudge the lifter slip off of the barcode and onto the microarray surface using fine forceps to allow hybridization to proceed (see Figure 3). Probe mixtures should be reacted for 3.5 hours at 42°C for gene expression applications, though different times and temperatures can be used for probe mixtures of different concentration and complexity. Probes derived from cloned and recombinant sources will probably produce satisfactory results within 5-10 minutes because the molecular complexity of such sources is low and H25K performance is extremely high. Probe mixtures derived from complex sources such as human genomic DNA, mRNA and protein may require more than 3.5 hours to produce strong signals. In rare cases, users may opt for up to 18 hour reaction times, though probe drying and elevated background noise can become an issue with prolonged hybridizations. After the hybridization step is complete, users should proceed quickly to the wash steps to prevent probe drying and elevated background.
4. Wash H25K to remove unbound probe molecules. After the probe binding reaction is complete, H25K should be washed to remove unbound material. In the case of H25K hybridization reactions, oligonucleotide-DNA or oligonucleotide-RNA hybrids are typically washed at high-stringency to remove labeled probe molecules that have bound weakly or non-specifically. Lesser stringency can be use in cases where DNA and mRNA probe mixtures are derived from homologous species such as primate, mouse and rat. For protein binding applications, oligonucleotide-protein complexes should be washed to remove proteins that have bound weakly or non-specifically. H25K oligonucleotides are bound in a highly stable manner to the glass substrate, and therefore the oligonucleotides themselves are stable to elevated temperature (≤70°C), low salt, and other neutral pH (pH 6-8) treatments. Users should avoid extremes in temperature and pH to avoid damaging the H25K oligonucleotides. Protein experiments with H25K typically make use of a series of room temperature (20-25°C) washes with phosphate-buffered saline (PBS) buffers containing dilute additions of detergent (e.g. 0.1-0.01% TritonX100). Prior to washing, the lifter slip must be removed from the H25K surface to allow efficient washing and to avoid scratching the H25K surface. For gene expression applications, quickly remove the lifter slip by submerging H25K for 5-10 sec in 42°C in Wash Buffer A and wash as follows: 5 min at 42°C in Wash Buffer A, 5 min at room temperature in Wash Buffer B, and 1 min at room temperature in Wash Buffer C, followed by a 1 sec dunk in dH2O. Washes should be performed in High-Throughput Wash Stations with moderate mixing or in the TrayMix™ S4 Hybridization Station. Users can experiment with different wash buffers and different temperatures (20-65°C) to optimize the washing process for a particular H25K application. Following the final wash step, H25K should be dried quickly by centrifugation for 3 sec in an Arrayit Microarray High-Speed Centrifuge in preparation for scanning.
5. Detect H25K signals by scanning or imaging. H25K should be inserted into a microarray scanning or imaging device to acquire the signals. The Arrayit InnoScan® 710 and 910 Fluorescence Scanners and the highly affordable Arrayit SpotLight™ Microarray Fluorescence Scanner works well for this application, though H25K is compatible with all major brands of microarray glass slide scanners including systems manufactured by PerkinElmer, Axon, Bio-Rad, Applied Precision, and others. Scanners and imagers should be capable of at least 10 µm resolution, the lowest recommended resolution setting for H25K. Scanning at 3-5 µm resolution produces higher quality H25K images and is recommended for most applications. The selected scan area should be 22 x 60 mm. In addition to the resolution and scan area settings, correct settings should be used for wavelength(s), gain, laser intensity, PMT, and other technical parameters required to produce high quality, data-rich images. Applications such as genotyping and mapping typically produce signal intensities spanning only 1-2 orders of magnitude, and in such cases a single set of instrument settings and a single image usually suffice to capture all of the H25K data. For expression analysis applications involving complex mixtures of mRNA varying greatly in concentration, multiple scanner settings (e.g. low and high sensitivity) across two or more images can be used to capture signals spanning up to 6 orders of magnitude. Multi-color experimentation involving, for example, cyanine 3 and cyanine 5 labels, obviously require data capture at two different wavelengths (e.g. 532 and 635 nm). H25K images should be saved as 16-bit TIFF files for quantification and downstream analysis. A 10 µm scan of H25K hybridized with 1.0 µg of Cy3-labeled cDNA probe derived from total human mRNA is shown in Figure 7.
6. Quantify the H25K signals. Arrayit MAPIX® quantification software is highly recommended for H25K quantification. Create a quantification sub-grid containing 48 subgrids of 24 x 24 elements. Place the subgrid over the H25K TIFF file, making sure that all subgrids and spots are properly aligned. MAPIX® quantification settings should be set to allow the software to find all of the printed spots in an automated manner. The following settings work well for most applications: Max position offset (% pitch) 30, Min. diameter (% Dth) 80, Max. diameter (% Dth) 190, S/B border width (pixels) 1.6 and Background diameter (pitch) 1.6. Once the grid has been accurately aligned, the quantification process should be initiated to extract data values from H25K using the photmetric calculation command. The extracted data should be saved and then opened in Microsoft Excel or some other software program for downstream analysis.
7. Perform data analysis. Properly quantified H25K images will yield data of extraordinary value. Values should be analyzed using a variety of inclusion and exclusion criteria to ensure statistical relevance of all values. For most applications, background-subtracted median signal intensities provide the most accurate reporting of spot signal intensity. Data from multiple images can be compared to identify up- and down-regulated genes in expression profiling experiments. Arrayit offers highly sophisticated Genome Pathway Builder™ software analysis for Biomarker Discovery and Validation Services customers. Genome Pathway Builder™ constructs biological pathways from vast quantities of expression data, providing customers with a detailed molecular understanding of a disease process. Genome Pathway Builder™ also allows pharmaceutical clients to understand the mode of drug action as well as off-targets as a means of benchmarking pharmaceutical leads prior to and during clinical trials.
Figure 4. H25K Human Genome Microarray orientation and location on the 25 x 76 mm glass substrate slide. The printed H25K microarray occupies 18 x 54 mm area (red) on the same side of the glass substrate slide as the barcode. Samples reacted with H25K, either by cover slip and through the use of an automated hybridization stations, must exceed the printed area shown to ensure coverage of all 26,304 printed elements. The 22 x 60 mm lifter slip included in the H25K Kit provides complete coverage of the printed area.
Figure 5. The TrayMix™ S4 Hybridization Station is highly recommended for automated hybridization and washing of H25K Human Genome Microarrays. Up to four H25K Human Genome Microarrays can be reacted simultaneously with one TrayMix™ S4, allowing 8 H25Ks to be run per instrument per day. One computer can be used to control up to 8 individual TrayMix™ S4 instruments for an H25K throughput of 64 microarrays per day. Users should follow the instructions carefully when using this instrument for automated H25K applications.
Figure 6. Arrayit High Throughput Wash Station for the H25K wash steps. Following the probe reaction and lifter slip removal, H25K can be washed using the High Throughput Wash Station (Cat. HTW) containing 400 ml of Wash Buffer A, B or C. A magnetic stir plate and stir bar provide moderate buffer agitation of 1-25 H25K microarrays per wash station.
Figure 7. Scanned image of a portion of H25K hybridized with with 2.0 µg of Cy3-labeled cDNA probe derived from total human mRNA. The intense signal strenght and ultra-low background permit users to quantify rare mRNAs not detectable on other platforms. Greater detectivity permits the identification of biomarkers for major human disease pathways.
Figure 8. Scanned single color image of the entire H25K microarray using 2.0 µg of Cy3-labeled cDNA probe derived from total human mRNA from human fibroblast cells. Genomic expression profiling on H25K is being used by Arrayit scientists to decipher the molecular basis of major human diseases.
Figure 9. H25K human genome expression profiling services using two-color labeling of a control and test sample to identify a differentially expressed gene serving as an expression biomarker for a major human disease.
Figure 10. Arrayit H25K Human Genome Chips provide credible $1,000 genomes for all researchers.
Please contact us if you have any questions, comments, suggestions, or if you need technical assistance. By electronic mail, use firstname.lastname@example.org and type "H25K technical assistance" into the subject line. By email, email@example.com between the hours of 8 AM and 8 PM PST Monday through Friday. We want to hear about your successes and are always happy to publish contributed H25K sample data on our website.
1. Weak signals
Degraded DNA, RNA or protein. Check sample integrity.