Arrayit has developed HybIt® Hybridization Solution containing an advanced mixture that improves microarray hybridization reactions by increasing signal intensities and reducing background in reactions with cDNAs and long oligonucleotides. Use HybIt® to produce superior data with microarrays printed on atomically flat Super Microarray Glass Substrate Slides.

Table of Contents
Introduction
Quality Control
Product Description
Technical Assistance
Short Protocol
Complete Protocol
Literature Cited
Requirements
Troubleshooting Tips
Ordering Information
Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your genomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use Arrayit HybIt® Hybridization Solution.

Quality Control
Arrayit assures the performance of this product. The finest scientific research went into the development of this product. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the ingredients conform to the highest industry standards.

Product Description
The Arrayit HybIt® Hybridization Solution is an advanced buffering system containing a patent-pending mixture of salts, detergents, accelerants, and stabilizing agents. HybIt® Hybridization Solution increases the efficiency and specificity of microarray hybridization reactions by expediting base pair formation between complementary target sequences attached to the microarray surface and labeled probe molecules in solution.  Users will appreciate the following features:

• Increases hybridization signals by accelerating reaction kinetics.
• Optimized for cDNAs and oligonucleotides.
• Increases detectivity (sensitivity) by reducing background fluorescence.
• Reduces surface tension enabling a uniform hybridization layer.
• Buffering components stabilize extended hybridizations.
• Compatible with many different surface chemistries.
• Arrives as a pre-mixed 1.25X solution that is sterile and ready to use.
• Costs <$1 dollar per hybridization reaction. 

Figure 1.  Oligonucleotide hybridization performed using HybIt® Hybridization Solution.  A total of 8,000 human cDNAs were purified with Arrayit’s PCR Purification Kit, dried to completion and resuspended in 4.0 µl dH20. A total of 4.0 µl of 2X MSS Plus was added to each sample and the samples were mixed thoroughly by pipetting. The samples were printed on SuperAmine Microarray Substrates, the printed substrates were processed according to the product instructions, and hybridized with a Cy3-labelled random 9-mer at 10 µM concentration in 1X HybIt® Hybridization Solution for 4 hours at room temperature (22°C).  The microarray was washed and scanned for fluorescence emission. The quality of the fluorescent signals are easily observed.

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). Please remember that we want to hear about your successes!

Short Protocol (Steps 1-8)
1. Obtain a nucleic acid microarray.
2. Process the microarray to prepare for the hybridization reaction.
3. Purify the hybrization probe using a suitable purification method.
4. Resuspend the probe in 1.0 µl dH20 and add 4.0 µl 1.25X HybIt® Hybridization Solution.
5. Hybridize the probe to the microarray.
6. Wash away the unbound probe (Arrayit Wash Buffers recommended).
7. Scan or image the microarray to acquire fluorescent signals.
8. Quantify the hybridization results and model the data.

Complete Protocol (Steps 1-8):
1. Obtain nucleic acid microarrays, either by manufacturing them or purchasing pre-made or custom microarrays. Microarrays can be printed using a suitable motion control technology equipped with Arrayit’s Stealth™ or ChipMaker™ Micro Spotting Technology or an equivalent device. Microarrayers can be purchased from a variety of sources including Virtek Vision (Ontario, Canada), Cartesian Technologies (Irvine, CA), Packard Biochip Technologies (Billerica, MA), GeneMachines (Belmont, CA), and many other providers. HybIt® is formulated to improve hybridization results with microarrays printed on a wide range of different microarray manufacturing systems.

2. Process the microarrays to prepare for the hybridization reaction.  When using SuperAldehyde Substrates, make certain to allow the printed substrates to dry overnight before processing. Drying can be carried out on the platen of the microarrayer, provided the humidity is <40%.  Printed substrates can also be dried in slide boxes, by keeping the lid slightly ajar overnight. Drying is required for Schiff’s base formation between the amino-lniked DNA to the SuperAldehyde surface.  Microarrays printed on SuperAmine Substrates can be used within 1 hour after printing, though the substrates must be baked at 80oC for 80 minutes or crosslinked with ultraviolet light (e.g. Stratagene Stratalinker) to strengthen the attachment of the DNA to the SuperAmine surface.  After proper drying and/or baking or U.V. crosslinking, the substrates should be processed to remove unbound DNA.  Many different protocols have been used successfully for substrate processing.  The SuperAldehyde and SuperAmine Substrate product documentation provides additional information on substrate processing.

3. Prior to hybridization, make certain to purify both fluorescent and non-fluorescent probes to remove contaminants that can lead to increased background. Probes can be purified with the ArrayIt Fluorescent Probe Purification Kit or an equivalent purification system.

4. The purified and dessicated probes should be resuspend in 1.0 µl dH20 and 4.0 µl of 1.25X HybIt® Hybridization Solution. Prior to use, make certain to pre-warm the HybIt® Hybridization Solution for 1 min at 65°C and mix the Solution by inverting the tube several times to make sure the components are mixed properly.  For a 20 µl hybridization volume, add 4.0 µl dH20 and 16.0 µl of 1.25X HybIt® Hybridization Solution, and scale the volumes accordingly for larger and smaller probe volumes.  Probe molecules can be a variety of fluorescent and non-fluorescent species including single-stranded DNA, RNA and oligonucleotides.

5. Hybridize the probe to the microarray.  Hybridization reactions can be performed using glass cover slips and 1.25 µl of probe solution per 1.0 cm2 of cover slip.  For best results, add the probe to one edge of the microarray surface, and gently lower the cover slip onto the microarray with fine forceps allowing the probe to sheet evenly across the surface between the cover slip and the microarray substrate. Pre-heating the probe solution to the hybridization temperature (e.g. 65°C) has been shown to reduce background fluorescence.  Once the cover slip is lowered onto the microarray, transfer the substrate with cover slip to a pre-warmed Hybridization Cassette containing 10.0-µl dH20 to maintain 100% humidity during the hybridization reaction.  A volume of 10 -20 ul dH20 placed under the substrate can also be used to further reduce sample dessication.  Seal the cassette and hybridize for 1-12 hrs at the proper temperature. For short oligonucleotides (e.g. 9-mers) a hybridization termperature of 22°C works well and for 15-mers, 42°C yields nice results. Long oligonucleotides and cDNAs are hybridized commonly at 65°C.

6. Wash the hybridized substrate to remove the unbound probe molecules.  Remove the microarray from the Hybridization Cassette and transfer the substrate immediately to an Arrayit High-Throughput Wash Station and wash the substrate with the appropriate buffers. Arrayit  Wash Buffers A, B and C are recommended for cDNA hybridizations and Arrayit Wash Buffers 1, 2 and 3 are recommended for oligonucleotide hybridizations. “Home-made” buffers will also work well for many applications. For indirect labeling procedures that require “staining steps”, microarrays should be stained prior to moving on to the detection step (Step 7).

7. Once the microarray is washed, it should be scanned or imaged to acquire fluorescent signals. Insert the substrate into the ScanArray™ 5000 (Packard BioChip Technologies). Compatible detection systems are also available from Virtek Vision (Ontario, Canada), Axon Instruments (Hayward, CA), Applied Precision (Issaquah, WA) and several other vendors. Scan the area of the substrate containing the microarray.  The scan area, excitation source, laser power and PMT settings should be adjusted with the ScanArray™ software to provide optimal signals. Laser and PMT settings should be chosen to give maximal unsaturated signal with minimal background fluorescence. Typically, laser and PMT settings of 70% and 60-80% respectively yield good results with the ScanArray™ 5000 for most hybridized microarrays.

8. Save the hybridization results and quantify the data. Upload the scanned image tiff file into the ImaGene software (BioDiscovery) or a suitable quantitation package and examine each feature on the microarray for fluorescence intensity. Additional manipulations and mining programs can be used to generate ratios, scatter plots, clusters and the like.

HybIt® References

• Oligonucleotide Microarray-Based Mutation Detection of the K-ras Gene in Colorectal Cancers with Use of Competitive DNA Hybridization: http://www.clinchem.org/cgi/content/full/50/9/1688
• Library on a slide for bacterial comparative genomics: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=394321
• STEC-EPEC Oligonucleotide Microarray: A New Tool for Typing Genetic Variants of the LEE Pathogenicity Island of Human and Animal Shiga Toxin–Producing Escherichia coli (STEC) and Enteropathogenic E. coli (EPEC) Strains: http://www.clinchem.org/cgi/content/abstract/52/2/192
• Development and Applications of a ß-Catenin Oligonucleotide Microarray: http://clincancerres.aacrjournals.org/cgi/content/full/9/8/2920

Literature Cited
1. Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler, J., Lockhart, D. J., Morris, M. S., Fodor, S. P. A. (1996) Accessing genetic information with high-density DNA arrays. Science 274: 610-614.

2. de Saizieu, A., Certa, U., Warrington, J., Gray, C., Keck, W., and Mous, J. (1998) Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nature Biotech. 16: 45-48.

3. A comprehensive set of nearly 1,000 microarray citations is available in an electronic format at http://arrayit.com/e-library.

Recommended Equipment and Reagents
Stealth™ or ChipMaker™ Micro Spotting Technology
High Throughput Wash Station
Microarray Wash Station
Fluorescent Probe Purification Kit
Micro Spotting Solution Plus
Micro Spotting Solution
Hybridization Cassettes
SuperAldehyde or SuperAmine Substrates

Troubleshooting Tips
High Background:

• Contaminated cDNAs or oligonucleotides used for printing.  For PCR products, use the PCR Purification Kits.
• Used conventional buffers instead of HybIt® Hybridization Solution
• Hybridization temperature too low
• Probes were not purified sufficiently to remove unincorporated fluors. Use the Fluorescent Probe Purification Kit.