HybIt Microarray Hybridization Solution (1 ml of 1.25X)

Arrayit HybIt Microarray Hybridization Solution (1 ml of 1.25X) is an advanced mixture of salts, detergents, accelerants and buffering components that improves the quality of microarray hybridization reactions by increasing signals and reducing background for a wide range of DNA microarray applications in research, life sciences, agriculture, pharmaceuticals and microarray testing.

Buffers and Solutions - HybIt Microarray Hybridization Solution to Increase the Efficiency of DNA Microarray Hybridization Reactions for Research, Life Sciences and Pharmaceutical Applications

Arrayit offers HybIt Microarray Hybridization Solution (1 ml of 1.25X) containing an advanced mixture of reagents to improve microarray hybridization reaction efficiency by increasing signal intensities and reducing background in complementary DNA (cDNA) and long oligonucleotide microarray reactions. Arrayit HybIt Microarray Hybridization Solution will produce superior data with microarrays printed on Arrayit microarray slides (1 x 25 x 76 mm) and other high-end surfaces.

Table of Contents
Introduction
Quality Control
Product Description
Short Protocol
Complete Protocol
Equipment Requirements
Troubleshooting Tips
Technical Assistance
Scientific Publications
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 Microarray Hybridization Solution.

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

Product Description
Arrayit HybIt Microarray Hybridization Solution is an advanced buffering system containing a proprietary mixture of salts, detergents, accelerants, and stabilizing agents. HybIt Microarray 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 including epoxy, amine and aldehyde
• Arrives as a ready-to-use pre-mixed 1.25X solution
• Affordable cost per hybridization reaction

Figure 1. Oligonucleotide hybridization performed using Arrayit HybIt Microarray Hybridization Solution (Cat. HHS). A total of 8,000 human cDNAs were purified with Arrayit’s PCR Purification Kit (Cat. PCR96-100), dried to completion, re-suspended in 4.0 µl dH20, and made print-ready by adding 4.0 µl of 2X MSS Plus (Cat. MSP) to each cDNA sample. The cDNAs samples were mixed thoroughly by pipetting and printed in triplicate on Arrayit SuperAmine Microarray Substrate Slides (Cat. SMM). The 24K printed slides were processed according to the SMM product instructions, and hybridized with a Cy3-labelled random 9-mer at 10 µM concentration in 1X HybIt® Microarray Hybridization Solution for 4 hours at room temperature (22°C). The microarray was washed and scanned for fluorescence emission. The quality of the fluorescent hybridization signals is easily observed in the scanned microarray data.

Short Protocol (Steps 1-8)
1. Print a cDNA or long oligonucleotide 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 Microarray Hybridization Solution.
5. Hybridize the probe to the printed microarray.
6. Wash away the unbound probe using Arrayit Wash Buffers.
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. Print a cDNA or long oligonucleotide microarray. Arrayit contact printing technology works well for this application. Microarrays may also be purchased from Arrayit or another suitable vendor. Microarrays can be printed using a suitable motion control technology equipped with Arrayit Stealth, 946 or professional microarray printing technology. Microarrayers can be purchased from a variety of suppliers including Arrayit and others. HybIt® is formulated to improve hybridization results with microarrays printed on a wide range of different microarray manufacturing systems and surfaces.

2. Process the microarrays to prepare for the hybridization reaction. When using Arrayit SuperAldehyde Substrate Slides, make certain to allow the printed substrates to dry overnight before processing. Drying can be carried out on the platen of the microarray printer if the relative humidity is ≤40%. Printed slides can also be dried in a vacuum oven or in slide boxes by keeping the lid slightly ajar overnight. Drying is required for Schiff’s base formation between the amino-linked DNA to the SuperAldehyde surface. Microarrays printed on SuperAmine Substrate Slides can be used within 1 hour after printing, though the slides must be baked at 80°C 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 slides should be processed to remove unbound DNA. Many different protocols have been used successfully for slide processing. Arrayit SuperAldehyde and SuperAmine Substrate Slides product documentation provides additional information on slide 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 (Cat. FPP) 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 Microarray Hybridization Solution. Prior to use, make certain to pre-warm the HybIt Microarray 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 Microarray 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 or using LifterSlips and 7 µl of probe solution per 1.0 cm2 of lifter 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 slide. Pre-heating the probe solution and the microarray slide to the hybridization temperature (e.g. 42°C or 65°C) has been shown to reduce background fluorescence considerably. Once the cover slip is lowered onto the microarray, transfer the slide with cover slip to a pre-warmed Hybridization Cassette containing 10.0 µl of dH20 to maintain 100% humidity during the hybridization reaction. A volume of 10-20 µl of dH20 placed under the slide can also be used to further reduce sample dessication. Seal the cassette and hybridize for 1-4 hrs at the proper temperature. For short oligonucleotides (e.g. 9-mers) a hybridization temperature 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 slide 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 (Cat. HTW) and wash the slide 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. 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 slide into the Arrayit InnoScan Microarray Scanner. Compatible scanners are also available from Molecular Devices. Scan the area of the slide containing the microarray. The scan area, excitation source, laser power and PMT settings should be adjusted with MAPIX® or the scanner controller software to provide optimal signals. Laser and PMT settings should be chosen to give maximal unsaturated signal with minimal background fluorescence.

8. Save the hybridization results and quantify the data. Upload the scanned image tiff file into MAPIX, ImaGene 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 other data representations.

Recommended Equipment and Reagents
Microarray printing technology
High Throughput Wash Stations
Microarray printers
Fluorescent Probe Purification Kits
Micro Spotting Solution Plus
Hybridization cassettes
Microarray slides

Troubleshooting Tips
High Background:

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