Arrayit eChips Services provide unprecedented access to microarray content. By uniting the power of microarray technology with the universality of the Internet, Arrayit makes microarrays affordable, versatile and easy to use. Every microarray is manufactured in a class 100 cleanroom using the most advanced robotics, surfaces and coupling chemistries in the industry. Our particle- and ribonuclease-free manufacturing laboratories provide microarrays of unsurpassed quality.  eChips allow customers to mine existing databases for sequences of interest and send them by electronic mail for microarraying. Synthesized oligonucleotides and peptides are printed on optically flat SuperAmine or SuperAldehyde substrates.  eChips allow researchers to create a nearly infinite variety of microarrays for basic research, clinical, pharmaceutical and agricultural applications.  Because the customer performs all of the steps downstream of manufacturing, the data remain completely confidential.  Imagine any nucleic acid or peptide molecule, from any organism, for any application on a pristine surface and you are beginning to understand the breadth and depth of our Custom Microarray Services. Arrayit eChips set a new standard in microarray technology.

Table of Contents

• Introduction
• Quality Control and Use
• Description of Services
• Technical Assistance
• Short Protocols
• Design Note
• Complete Protocols
• Equipment Requirements
• Publications
• Tips for Design and Use
• Product Contents
• Ordering Information
• Storage Conditions
• Warranty

Introduction
Congratulations on taking a big step towards improving the affordability, quality and speed of your genomics, biomedical, pharmaceutical and agricultural research. This booklet contains all the information required to take full advantage of Arrayit Corporation eChips.

Quality Control and Use
Arrayit Corporation takes every measure to assure the quality of our eChips.  The finest microarray cleanroom technology was used to develop these products. Rigorous quality control monitoring on a chip-by-chip basis guarantees that each microarray biochip conforms to the highest industry standards. Please be aware that certain types and uses of microarrays may be patent protected. Arrayit Corporation does not endorse the improper use of its Custom Microarray Services.  Please consult your intellectual property expert to assure that your particular application is licensed properly.

Description of Services
eChips from Arrayit Corporation provide a virtually unlimited variety of microarray biochips at an affordable price. All of our microarrays are fabricated in a state-of-the-art class 100 cleanroom, with 0.1 µm filtered air and precise temperature and humidity control.  This nearly particle-free, sterile setting provides a custom microarray manufacturing environment that rivals facilities in the semiconductor industry.  Users will appreciate the following product features:

eChips: Customer provides information for up to 9,600 sequences

• Pristine, optically flat glass printing substrates
• Proprietary, covalent coupling chemistries that are stable to 100°C
• Substrates (25 x 76 x 0.96 mm) fit all commercial microarray detection systems
• Substrate corner chamfer enables unambiguous slide orientation
• Barcoding allows automated chip identification
• Any sequence, any organism, any application
• Arrays of DNA, RNA and peptides
• Utilize a wide range of parallel analysis applications
• Examine both hybridization and protein interactions
• State-of-the-art class 100 cleanroom sets a new standard for microarray fabrication
• Robotics with ± 1 µm positional accuracy
• Triplicate spotting assures high data precision and low CVs
• Low reaction volumes (2-30 µl) speed kinetics and maximize signals
• Arrayit's Patented Printing Technology assures highest microarray quality
• Total confidentiality of all results!
• Develop new assays, publish, discover and patent results
• Internet savvy services leverage electronic design and ordering
• Reasonable turnaround time of 2-6 weeks (larger orders for oligo design, synthesis and arraying may take longer)

Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance.  By electronic mail, arrayit@arrayit.com (under the subject heading please type "ArrayIt technical assistance").  By telephone: (408) 744-1331, Monday–Friday PST 8:00 am – 6:00 pm. Please remember that we want to hear about your successes!

Short Protocols
1. Contact Arrayit Corporation with your project information to obtain a quote.
2. Email ID numbers or sequences by email to arrayit@arrayit.com as Excel or tab delimited text file. 
3. Place order based on quote, wait 1-4 weeks depending on the details of your order.
4. Obtain eChips by UPS shipping.

Table 1. Synthetic sequences for eChips.

Target	Assay	Sequence Length (nt)	5’ Modification	Comments
DNA	Sequencing by Hybridization	5-15 mer	C6- or 12-amino modifier recommended	Can add fluorescent moieties at 3' end
DNA	Genotyping	10-25 mer	C6- or 12-amino modifier recommended
DNA	Gene Expression	50-70	C6- or 12-amino modifier recommended	50 nt is upper limit of mass spec for QC of oligos
DNA	Mapping or Contig Analysis	50-125	C6- or 12-amino modifier recommended
RNA	All	Up to 35 bases	C6- or 12-amino modifier recommended	Can add fluorescent moieties at 3' end
Peptide	All	1-35 residues	No modification required but Amino Modification is recommended for best results.

Design Note
If you are designing your own oligonucleotide target sequences, we recommend Array Designer by Premier Biosoft as a convenient and powerful software package. If you are using a different program, we Hhighly recommended using the "blastn" program and the "nr" database, allowing the user to confirm the sequence and check for homologous sequences in the human and other genomes for people making oligos chips (http://www.ncbi.nlm.nih.gov/BLAST/).Users should use the "blastn" program and the "nr" database. BLAST will crunch an oligo against the entire database, allowing the user to confirm the sequence and check for homologous sequences.

Complete Protocols
1. Contact Arrayit Corporation with your project information to obtain a quote. Our eChip services are divided into three components:  oligonucleotide or peptide design, oligonucleotide or peptide synthesis, and custom microarray printing.  Contact arrayit@arrayit.com with your project information so that we can provide you with a quote.  Our staff of highly trained scientists and engineers can provide design, content and application assistance if you have any questions. The cost per chip depends primarily on the number of chips ordered and the number of elements printed. There are additional charges for oligonucleotide and peptide design and synthesis. For example, an order of 192 sequences (1/2 of a 384 well plate) printed on 25 chips would cost $159 per chip or $3975, plus the additional cost of synthesis and design. The cost of the synthetic sequences is a one-time charge for as many chips as can be made with the synthesis (typically ~3,000 chips). We can also design your oligonucleotides and peptides for a cost of $20.00 per sequence.

2. Send a Purchase Order number, referencing your quote number to: Arrayit, 524 East Weddell Drive, Sunnyvale, CA 94089.  Phone: (408) 744-1331, Fax (408) 744-1711 or by email to arrayit@arrayit.com.

3. Send sequences by electronic mail as Excel or tab delimited text file.  Send the Excel spreadsheet or tab delimited text file as an attachment to an email that identifies the order and has complete contact information.  Please remember that due to a high volume of orders, all electronic mail communications must contain complete contact information, including name, affiliation, telephone number and FAX number.  Failure to provide these details may delay your order.  Your oligo sequences or genomic information for oligo design can also be sent to us by overnight mail in an Excel spreadsheet or tab delimited file format a 3.5 inch floppy disks or Zip disks (PC formatted) at the address above. Please include a letter or email that references your quote number and has complete contact information.

4. Wait 2-6 weeks depending on details of your order.  Once your order is placed, we will provide a more exact timeline. The time to process orders depends on the volume of orders in the queue and on the size of your order. Arrayit Corporation will make every attempt to process your order as quickly as possible.

5. Obtain eChips shipped directly to you by Express Mail or by the shipping method that you specify. All eChips will arrive complete with barcodes and corner chamfer.  The barcode contains an identifying number that is unique to each chip in your order.  The corner chamfer is provided to allow the user to determine easily which side of the substrate contains the printed microarray and defines the top of the substrate.  The printed microarray is located on the same side of the substrate as the barcode.  The corner chamfer should appear in the upper right corner if the barcode is at the bottom of the substrate. You will also receive a content file that shows the location of each printed target sequence on the microarray substrate.

6. Process the eChips to remove unbound target sequences.  Processing protocols vary according to the Substrate used and the target sequences.  Detailed protocols are available electronically on the corresponding product page.

7. Label and purify probes.  There are a myriad of enzymatic and synthetic methods used to label probes derived from RNA, oligonucleotides and proteins. Samples can be labeled using direct labeling whereby fluorescent tags are incorporated into the molecules in the sample. Alternatively, samples can be labeled by indirect means prior to hybridization such that epitopes such as biotin or amino allyl are incorporated into the sample, then the molecules are made fluorescent using fluorescently labeled streptavidin or reactive fluorescent dyes.  Post hybridization detection can also be achieved using secondary labeling molecules such as dendrimers (Genisphere, Inc) or antibody-containing fluorescent molecules. Samples can be nucleic acids, proteins or other types of molecules.  Users should select the labeling procedure that works best for their application.  Labeling procedures and tips are available under Super Microarray Substrates, Hybridization Products, Amplification and Labeling, and Tools.  Our specialists are happy to provide advice and feedback on probe labeling and purification.

8. Perform a hybridization or biochemical reaction.  Probes should be reacted with the processed eChips to allow the molecules to interact with their cognate targets on the chip.  This step is best accomplished by reacting the sample to the processed microarray underneath a cover slip positioned over the microarray.  The cover slip should extend ~2 mm beyond the edges of the microarray on all sides to ensure that the sample covers all the spots. Sample volumes are typically ~2.0 µl per cm2, such that total reaction volumes of 3.0 µl and 20 µl work well for the 12 mm x 12 mm and 22 mm x 40 mm cover slips, respectively. The corner chamfer should be at the upper right side of the substrate relative to the user when the microarray is held vertically (with the barcode at the bottom). Buffers suitable for nucleic acid hybridizations include 5X SSC + 0.2% SDS, 1X UniHyb™and 1X HybIt®.  Other types of buffers such as those containing SSC, SSPE, SDS, and TritonX-100 have also been used successfully.  Sample is best added to the microarray by placing the drop at the edge of the cover slip and allowing the sample to spread out between the microarray and the cover slip by capillary action. Once the sample is added, immediately place the microarray in an enclosed chamber such as a Hybridization Cassette and bring the reaction up to temperature as quickly as possible. It is critical to prevent desiccation during the biochemical reactions, as drying is the major source of elevated background fluorescence in microarray experiments. Desiccation is prevented by adding 5-25 µl of buffer to the reaction chamber.  Reaction temperatures are typically 37-65°C, though different experiments may require higher or lower reaction temperatures.  Reaction times are usually 2-12 hours.  Information on hybridization reactions can be obtained under Hybridization Products, Super Microarray Substrates, and DNA Microarray Protocols.  Our specialists are happy to provide advice and feedback on hybridization, protein, and other types of biochemical reactions involving eChips.

9. Wash eChips to remove unbound probe molecules.  Once the eChips are reacted with labeled probe samples, the chips must be washed to remove unbound molecules. Following the biochemical reaction, unbound sample should be washed off the surface. For cDNA hybridizations, the following protocol works well. Remove the microarray from the Hybridization Cassettes and place it in a High-Throughput Wash Station and wash according to the protocols provided with the Microarray Wash Buffers.  Wash conditions should be modified for different types of reactions and different applications.  Make sure the cover slip falls off within 1 minute after transfer into the wash buffer.  Wash buffers pre-heated to 42°C can be used to speed the release of the cover slip from the microarray.  Failure to remove the cover slip within 1 minute after transfer to the wash buffer may lead to elevated background fluorescence. There are numerous washing protocols available and details are available under DNA Microarray Protocols, Super Microarray Substrates, Hybridization Products, and Microarray Wash Buffers.  An electronic library containing nearly 1,000 publications provide a wealth of information on microarray wash procedures. Spin the substrates dry by centrifugation for 10 seconds in a Microarray High-Speed Centrifuge.  Our specialists are happy to provide advice and feedback on eChip washing strategies.

10. Detect the fluorescent signals.  Once the eChips are reacted and/or stained, and washed, the fluorescent signals must be detected.  Detection is best performed using one of the high-quality commercial instruments available.  Commercial systems are available from PE (Packard Bioscience), Axon, Applied Precision and several other vendors. Detection should be performed such that the most intense signals on the microarray produce sub-saturating values. Rare species in the probe mixture can be quantified using instrument settings that maximize detectivity. Adjusting the laser power, PMT gain, capture time and other parameters provide enormous detectivity in microarray assays.

11. Quantify and model the data. Data quantification and mining can be performed using a variety of custom and commercial software packages.

Tips for Design and Use
Table 2. A virtually unlimited number of different eChips are possible. Different eChips allow the user to explore diverse molecular interactions and applications.

Sequence Type	Interaction	Application
DNA oligonucleotide	DNA-DNA, DNA-RNA, DNA-protein, DNA-enzyme	enzymology, evolution, gene expression, genotyping, hybridization, mapping, SNP detection, structure-function, two hybrid analysis
Peptide	Protein-DNA, protein-RNA, protein-protein, protein-small molecule
RNA oligonucleotide	RNA-DNA, RNA-RNA, RNA-protein, RNA-enzyme	enzymology, evolution, gene expression, genotyping, hybridization, mapping, SNP detection, structure-function, two hybrid analysis

Product Contents
eChips are manufactured and packaged in a class 100 cleanroom setting, bar-coded and shipped in a rigid substrate box enclosed in sealed plastic shipping envelope. To open the eChips box, push downward firmly on the top of the box and pull gently upward on the packaging cloth.

Our eChip services are divided into three stages, Oligo Design, Oligo synthesis and Custom Microarray Printing.

Oligo Design

Oligo Design services are available at a cost of $10 per oligo. Our oligos are designed using our Arrayit Oligo Design Directive. This Directive ensures that the oligos:

• are mapped to within 1,000 nucleotides of the 3’ end of the selected
• are selected to avoid sequence repeats and long stretches of polyA, G, C and T
• melting temperatures of the oligos are matched within a selected range;
• checked against gene sequence databases to avoid cross-reactivity with transcript species of <70% sequence identity.

Oligo design services can take 1-2 weeks depending on the number of oligos required. Alternatively, you can design your own oligos using Array Designer Software from Premier Biosoft.

Oligo Synthesis
Arrayit oligos are available in 96 or 384 well formats or you may order your oligos through your own oligo synthesis provider. Oligo synthesis typically requires an additional 1-2 weeks, depending on the number of oligos ordered.

Peptide Synthesis
Arrayit peptides are available in 96 or 384 well formats or you may order your peptides through your own oligo synthesis provider. Peptide synthesis typically requires an additional 1-2 weeks, depending on the number of peptides ordered.

Custom Microarray Printing
Total custom microarray printing costs depend on the number of elements printed per microarray and the number of microarrays requested.  Prices per microarray are indicated on the table below.

Scientific Publications
Click here and here for recent scientific publications using ArrayIt® brand microarray products from Arrayit Corporation for custom microarray experimentation.

Recommended Equipment and Reagents
NanoPrint™ 2 Microarrayers
SpotBot® 4 Personal Microarrayers
InnoScan® Microarray Scanners
SpotLight™ 2 Microarray Scanners
SuperMicroarray Substrates
Microarray Hybridization Cassettes
High Throughput Wash Station
Microarray High-Speed Centrifuge
Protein Printing Buffer
BlockIt Blocking Buffer
Microarray Air Jet
Microarray Cleanroom Wipes
PCR Purification Kits
BlockIt Blocking Buffer
Micro-Total RNA Extraction Kit
MiniAmp mRNA Amplification Kit
Indirect Amino Allyl Fluorescent Labeling Kit
Universal Reference mRNA
Green540 and Red640 Reactive Fluorescent Dyes
Hybridization Buffers