HybIt 2 Hybridization Solution (1 ml of 1.25X)

Arrayit HybIt 2 Hybridization Solution, 1 ml of 1.25X is a formamide-based hybridization mixture of solvents, salts, detergents, buffering components and accelerants designed to increase the efficiency of long oligonucleotide, complementary DNA (cDNA) and genomic DNA microarray hybridization reactions for DNA microarrays applications including gene expression, genotyping, comparative genomic hybridization (CGH) and others. Arrayit HHS2 increases microarray signal strength and reduces background for improved signal-to-noise ratios and data precision, ships as 1.0 ml of 1.25X solution.

Buffers and Solutions - Hybridization Buffers - HybIt 2 Hybridization Solution for Long Oligonucleotide, cDNA and Genomic DNA Hybridization for Gene Expression, Genotyping and CGH DNA Microarrays

Arrayit offers HybIt 2 Hybridization Solutions containing an advanced formamide mixture that improves microarray hybridization reactions by increasing signal intensities and reducing background in reactions with cDNAs and long oligonucleotides. Arrayit HybIt 2 produces superior data for gene expression, genotyping, CGH and other DNA microarray applications. Arrayit HybIt 2 Automation Formulation is highly recommended for use with TrayMix™ Hybridization Stations and other automation instrumentation.

Table of Contents
Introduction
Quality Control
Product Description
Short Protocol
Complete Protocol
Equipment Requirements
Troubleshooting Tips
Technical Support
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 2 Hybridization Solutions.

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

Product Description
Arrayit HybIt 2 Hybridization Solutions are advanced hybridization solutions containing a proprietary mixture of solvents, salts, detergents and buffering components. Arrayit HybIt 2 Hybridization Solutions will increase the quality of microarray biochip hybridizations reactions involving base pairing interactions between complementary nucleic acid chains. Users will appreciate the following features:

• Increases signal by accelerating hybridization kinetics
• Increases sensitivity by reducing background fluorescence
• Reduces surface tension to provide a uniform hybridization layer
• Buffering components stabilize extended reaction times
• Compatible with many glass-based surface chemistries
• Arrives pre-mixed and sterile, no preparation required
• Affordable and convenient

Short Protocol (Steps 1-7)
1. Print microarrays or obtain Arrayit Custom printed microarrays.
2. Process the microarrays for hybridization.
3. Purify probe using ArrayIt Fluorescent Probe Purification Kit.
3. Resuspend fluorescent probe in 1.0 part dH20 and 4.0 parts HybIt 2 Solution.
3. Hybridize the probe mixture to the microarray under the appropriate conditions.
5. Wash away the unbound fluorescent probe. ArrayIt wash buffers A, B and C are recommended.
6. Scan the microarray for fluorescent signal.
7. Quantify the hybridization data.

Complete Protocol (Steps 1-7)
1. Print microarrays with an Arrayit microarray printing device or a suitable microarray technology. In principle, HybIt 2 should improve detection on many different microarray platforms.

2. Process the microarrays for hybridization. After the microarrays are printed, the slides should be allowed to dry at room temperature overnight for microarrays printed on SuperAldehyde, SuperAmine of SuperEpoxy slides. This can be accomplished by placing the slides in a slide box with the lid slightly ajar. Drying increases the binding of the DNA molecules including amino-modified DNA to the aldehyde and epoxy surfaces, as well as to amine chemistry. After drying, the slides should be processed to remove unbound DNA. Many protocols have been used for slide processing. Microarrays printed on SuperAmine should be crosslinked by either baking at 80°C for 80 minutes or by using a UV Crosslinker (120 mJ).

3. Purify the fluorescent probe mixture with the ArrayIt Fluorescent Probe Purification Kit and resuspend in HybIt 2 Hybridization Solution, which is provided as a 1.25X solution. This is accomplished by first resuspending the probe in 1.0 part dH20, and then adding 4.0 parts of HybIt 2 Hybridization Solution. Prior to using HybIt 2 Hybridization Solution, pre-warm the solution for 30 sec at 42°C and mix by inverting the tube (Cat. HHS2) or bottle (Cat. HHS2A) several times. A fluorescent probe desiccated to dryness would be re-suspended by adding 2.0 µl of dH20, followed by 8.0 µl of pre-warmed HybIt 2 Hybridization Solution. Probes should be single-stranded DNA or RNA molecules made by either reverse transcription or in vitro transcription of cRNA.

4. Hybridize the probe to the microarray under the appropriate conditions (42°C). This is accomplished by using 1.25 µl of probe in 1X HybIt 2 per cm2 glass cover slip. For best results, add the probe to one edge of the cover slip surface, and then 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 slide. Transfer the substrate slide with cover slip to a pre-warmed Arrayit Hybridization Cassette containing 10 µl of dH20 in the humidification reservoir. Seal the cassette and hybridize 1-4 hours at 42°C.

5. Wash away the unbound fluorescent probe. Remove the microarray from the hybridization cassette and immediately transfer the slide to an Arrayit Wash Station and wash according to directions.

6. Scan the microarray for fluorescent signals. Insert the substrate slide into the Arrayit InnoScan microarray scanner or a compatible detection system and scan the area of the slide containing the microarray. The scan area, excitation source, laser power and PMT settings can all be adjusted with the MAPIX® software. Laser and PMT settings should be chosen to give maximal unsaturated signal with minimal background fluorescence. Typically, laser and PMT settings of low and 10-100% respectively yield good results with the Arrayit InnoScan 710 and 910 microarray scanners.

7. Score the hybridization results. Upload the 16-bit scanned image tiff file into the MAPIX® software or a suitable quantitation package and examine each feature for fluorescence intensity. The data can be mined and modeled using additional software applications.

Equipment Requirements
Microarray pins and printheads
Microarray wash stations
Microarray purification kits
Microarray print buffers
Microarray hybridization cassettes
Microarray slides

Troubleshooting Tips
High Background

• Impure cDNAs or oligonucleotides used for printing
• Sub-optimal hybridization buffer (use HybIt 2 Hybridization Solution)
• Incorrect hybridization temperature
• Unincorporated fluors were not sufficiently purified away from probe (use Fluorescent Probe Purification Kits)