Arrayit Hybridization Cassette, Amethyst

Arrayit Hybridization Cassette, Black enables DNA and protein microarray coverslip-based hybridization reactions and features an anodized alloy hybridization chamber with a capacity of 1 standard microarray substrate slide (1 x 25 x 76 mm). The chamber consists of a durable transparent top with four thumb screws that allow assembly to the base, which holds the slide and contains a humidification reservoir to prevent evaporation during the hybridization reaction. The cassette base contains a 1.5 mm chamber depth and an elastomeric gasket for compression-based air-tight sealing with the top, one cassette per order, color amethyst.

Tools - Hybridization Cassettes - Hybridization Cassettes for Cover Slip-Based DNA and Protein Microarray Hybridization Reactions using Digital Water Baths and Laboratory Incubators

Arrayit hybridization cassettes for all standard glass substrate slide microarrays (1 x 25 x 76 mm), providing pristine hybridization chambers for cover slip reactions of DNA, protein and other types of microarrays. Compatible with all standard microarrayers, microarray printers, microarray spotters and microarray scanners, Arrayit hybridization cassettes are essential equipment for every microarray laboratory. Choose from single, five-place and extra deep configurations in six colors for ease of use.

Table of Contents
Introduction
Quality Control
Product Description
Short Protocol
Complete Protocol
Recommended Equipment
Technical Assistance
Troubleshooting Tips
Scientific Publications
Ordering Information
Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your life sciences, genomics, proteomics and microarray testing research. This booklet contains a complete set of protocols outlining the steps and principles needed to use Arrayit Hybridization Cassettes. This product has been designed for hybridization reactions involving DNA, protein and other types of glass substrate slide (1 x 25 x 76 mm) microarrays.

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

Product Description
Arrayit Hybridization Cassettes are designed for hybridization reactions involving cover slip reactions with DNA, protein, carbohydrate and other glass substrate slide (1 x 25 x 76 mm) microarrays. Users will appreciate the following features:

• Supports all standard glass substrate slide microarrays (1 x 25 x 76 mm).
• Single position, 5-place and extra deep designs in six colors.
• Water-tight and submersible into water baths for precise temperature control.
• Design allows stacking in a water bath to ensure uniform reaction temperatures among multiple cassettes.
• Wide range of reaction temperatures possible (0-70°C).
• Slot in cassette base allows easy disassembly and removal of microarray slides post-hybridization.
• Rugged design ensures durability.
• Inert construction materials provide pristine reaction environment and no out-gassing.
• Compact size: 2.1 x 5.7 x 10.2 cm.
• Made of chemically-resistant materials that provide highly affordable use.
• Clear top allows visualization during reactions.
• Easy to open and close by hand (no tools required).

Figure 1. Hybridization Cassette Plus (Cat. AHCP) holds five standard glass substrate slides (1 x 25 x 76 mm) and has a standard 1.5 mm chamber depth. The AHCPXD has 2.5 mm extra deep chambers suitable for sandwich hybridizations and other reactions involving thick cover slips.

Figure 2. Improved hybridization results with probe pre-heating and BSA addition. Shown are two microarrays of PCR products printed on SuperAldehyde Substrate Slides (Arrayit) with Stealth microarray printing pins (SMP3B) and a Cartesian 5500 robot. Printed microarrays were washed to remove unbound material and the double-stranded DNAs were denatured by boiling for 3 min in distilled water. The microarrays were hybridized in hybridization cassettes (Arrayit) for 5 hrs at 42°C under 18 mm x 18 mm optically flat cover slips with 5.0 µl probe solution containing 5X SSC + 0.2% SDS + 0.2 mg/ml bovine serum albumin (BSA) + 2 µM 15-mer oligonucleotide. The 15-mer oligonucleotide contained a Cy3 label on the 5' end. Hybridized microarrays were washed twice for 5 min each in 2X SSC + 0.2% SDS at 25°C, once for 1 min in 2X SSC at 25°C and spun dry for 1 min at 500 x g. Microarrays were scanned at 100% photomultiplier tube (PMT) and 100% laser settings with a ScanArray 3000 (Packard Instruments). (Left) Probe solution at 25°C applied to microarray and (Right) Probe solution pre-heated to 42°C and applied IMMEDIATELY to microarray. Improved results are easily observed in the right image compared to left image, including stronger signals, reduced background and increased uniformity. Probe pre-heating and BSA addition can both improve microarray data.

Short Protocol
1. Rinse Arrayit Hybridization Cassette with distilled water and dry thoroughly.
2. Make sure flexible rubber gasket is seated evenly in the cassette base gasket channel.
3. Add 50 µl of distilled water to the humidification reservoir located in the cassette base.
4. Insert the microarray slide (1 x 25 x 76 mm) into cassette chamber, DNA side up.
5. Aliquot the PRE-HEATED probe mixture onto the substrate slide at 2.0 µl solution per cm2 microarray area.
6. Gently lower the cover slip onto the microarray surface (thin forceps).
7. Quickly place the clear plastic cassette lid on top of the cassette chamber.
8. Apply gentle downward pressure and manually tighten (clockwise) the four thumb screws.
9. Check all four screws again to confirm a tight assembly.
10. Submerge the hybridization cassette into a water bath (0°-70°C). Clean distilled water is recommended for this application. The use of de-ionized water or additives such as EDTA can lead to corrosion of the hybridization cassette parts.
11. Allow the hybridization reaction to proceed (0.5-24 hrs).
12. After hybridization, remove cassette from the water bath and blot dry (polyester cleanroom wipes).
13. Manually loosen the four sealing screws (counterclockwise) and remove lid.
14. Remove the microarray slide from the cassette chamber (forceps).
15. Quickly transfer the slide to a beaker containing Wash Buffer A (cover slip will detach).
16. Wash away unhybridized sample with Wash Buffers A-C (~10 min total) and scan.

Complete Protocol
1. Rinse the Arrayit Hybridization Cassette chamber and lid with distilled water, making sure that salt crystals and other contaminants have been removed. Nitrile gloves must be worn at all times during this process. Hand oils, nucleases and other contaminants can interfere with the hybridization reaction, which proceeds in a ~10 µm layer between the microarray slide and the cover slip. After rinsing, dry the cassette chamber and lid thoroughly with polyester cleanroom wipes (Cat. MCW).

2. Prior to use, make sure that the flexible rubber gasket is seated evenly in gasket channel located around the perimeter of the cassette base. The gasket occasionally pulls free of the cassette chamber during use. If this occurs, re-insert the gasket into the gasket channel by applying light pressure. The gasket must be evenly seated in the gasket chamber to prevent leaks between the chamber and lid. Never attempt to use the hybridization cassette without a properly seated gasket.

3. Before inserting the microarray slide or substrate (1 x 25 x 76 mm) into the cassette chamber, add 50 µl of water (dH2O) into the humidification reservoir (groove) located in the base of the cassette chamber. The lower of the two grooves is recommended. The 50 µl of water evaporates during the hybridization reaction, producing conditions of 100% humidity in the cassette chamber. A humid environment prevents evaporation of the hybridization solution between the microarray slide and cover slip during the hybridization reaction. Failure to add 50 µl of distilled water to the humidification reservoir can cause drying of the fluorescent sample onto the microarray surface, leading to elevated non-specific fluorescence.

4. After adding 50 µl of water into the cassette chamber humidification reservoir, insert the microarray substrate slide (1 x 25 x 76 mm) with the reactive (DNA side) of the microarray facing upwards. Make sure that the slide is seated evenly on the base of the cassette.

5. Aliquot the fluorescent sample (probe) onto the slide at a volume of 2.0 µl of fluorescent probe per cm2 of printed microarray area. Arrayit substrate slides contain a corner chamfer that should in the upper right corner of the cassette for correct side orientation. Place the probe mixture directly adjacent to the printed microarray, rather than onto the printed microarray to avoid elevated background. Probe mixtures pre-heated to the hybridization temperature (42-65°C) and pipetted IMMEDIATELY onto the substrate slide produce lower and more uniform background. The cover slip should be added IMMEDIATELY onto the probe droplet and the mixture should sheet under the cover slip RAPIDLY (several seconds) for best results. The size of the cover slip should exceed the hybridization region of interest by at least 4 millimeters in each dimension. If the hybridization region is 1.8 x 3.6 cm, the corresponding cover slip should be 2.2 x 4.4 cm. A 2.2 x 4.4 cm cover slip requires 19.4 µl of hybridization solution. Standard hybridization buffers for microarray experiments contain 5X SSC or 6X SSPE and 0.2% SDS. The addition of 0.2 mg/ml BSA (Worthington) to the hybridization mixture can reduce background. For best results use hybridization buffers made by Arrayit. Prior to use, cover slips should be cleaned thoroughly with mild detergent and rinsed extensively with distilled water. Sonication for 5 min in distilled water and the use of optically flat cover slips from Arrayit also improve hybridization results. Cleaning is necessary to remove oils and other contaminants that are often present on commercial cover slips. After washing and rinsing, cover slips should be dried thoroughly with polyester cleanroom wipes (Cat. MCW) and inspected to make sure they are clean and free of dust and other debris. Polyester cleanroom wipes give superior results to paper wipes and should be used.

6. To initiate a hybridization reaction, gently lower the cover slip containing the fluorescent sample onto the microarray surface, using a pair of fine forceps. For right-handed persons, lower the cover slip containing the sample from left to right. For rectangular cover slips, left to right will correspond to the narrower of the two dimensions. Gently lowering the cover slip causes most air bubbles to exit naturally. The use of sonication-cleaned cover slips, optically flat cover slips, and pre-heated samples (42-65°C) all improve the quality of hybridized microarrays. The sample should sheet RAPIDLY under the cover slip (a few seconds) for best results. In certain cases, small air bubbles can become trapped between the microarray substrate and the cover slip. If this occurs, do not attempt to move the cover slip because most air bubbles exit the slide/cover slip interface after several minutes of hybridization.

7. The standard length of time for hybridization reactions involving 100 nucleotide probe and target pairs is 1-4 hrs at 62°C. Hybridization reactions involving short oligomers (15-25 bases) can be performed for 0.5-2 hrs at 37°C-42°C. To some extent, hybridization times and temperatures can both be adjusted for specific research applications. Once the cover slip is placed onto the microarray surface, quickly place the clear plastic cassette lid on top of the cassette chamber so that the four sealing screws align with the threaded holes in the cassette base.

8. Once the cassette lid is positioned correctly on top of the cassette base, manually tighten each of the four sealing screws by applying gentle downward pressure and turning the screws in a clockwise manner until turning becomes difficult (3-4 half turns). Check to see that the rubber sealing gasket is seated correctly in the gasket groove. If the gasket is unevenly seated, remove the lid and re-position the sealing gasket before tightening the four sealing screws.

9. After all four sealing screws have been manually tightened, double check all four screws by turning each clockwise again to make sure that the cassette lid is firmly sealed against the rubber sealing gasket in the cassette base. Screws should be finger tight. Do not tighten excessively. Use of tools such as pliers are not required and can permanently damage the hybridization cassette.

10. Once the hybridization cassette containing the microarray is sealed properly, submerge the cassette into a water bath incubator set at the desired temperature. Once a microarray is placed inside the hybridization cassette, do not invert the Hybridization Cassette at any time before, during or after the hybridization reaction. Always keep the cassette (clear lid) facing upwards. Inverting the cassette can cause the microarray slide and cover slip to adhere to the underside of the cassette lid, leading to a loss of hybridization sample and poor results. Arrayit hybridization cassettes are designed for a wide range of hybridization temperatures (0°-70°C), although 37°C-70°C is suitable for most hybridization reactions. Experiments involving multiple hybridization cassettes should be set up sequentially (i.e. one at a time). Arrayit hybridization cassettes are carefully designed to allow easy stacking. Water circulation between cassettes makes it easy to achieve uniform reaction temperatures. Most commercial water bath incubators can accommodate 10-20 Arrayit hybridization cassettes for high-throughput applications in a compact space.

11. Incubate the Hybridization Cassette with the clear cassette lid facing upwards in the water bath incubator for the desired period of time. Typically, 6-12 hr at 37°C-65°C is suitable for most applications. Abundant fluorescent species have been detected after hybridization times as short as 5 min. ArrayIt® hybridization cassettes are tested to withstand 72 hr at 70°C.

12. Following the hybridization reaction, remove the Arrayit hybridization cassette from the water bath incubator and dry the outside of the cassette by blotting briefly with paper towels. Removing excess water from the outside of the cassette prevents incubator water from flowing into the cassette chamber once the four sealing screws are loosened.

13. Place the Arrayit hybridization cassette on the laboratory bench and manually loosen the four sealing screws by turning each of them in a counterclockwise direction. When the four sealing screws have been loosened completely (3-4 half turns), remove the clear cassette lid. Under certain circumstances, a slight vacuum will prevent the manual removal of the cassette lid. If this occurs, insert a forceps into the slot at the base of the chamber and apply gentle upward twisting pressure.

14. Place the lid aside and remove the microarray slide from the cassette chamber. Under certain circumstances, the slide will adhere to the base of the cassette. If this happens, insert a forceps into one of the grooves in the cassette base and detach the slide from the base by applying gentle upward pressure.

15. Quickly transfer the microarray slides to a High Throughput Microarray Wash Station, containing 1X Wash Buffer A. A beaker with a 6-substrate slide wash station also works well for this application. For hybridizations involving probe and targets pairs of >100 bp, 1X Wash Buffer A at 25°C for 5 min works well to remove most of the un-hybridized material. Gentle agitation in 1X Wash Buffer A will cause the cover slip to float free from the microarray surface, 10-30 sec after submerging the microarray into the wash buffer. If the cover slip does not float free from microarray surface, gentle pressure with a fine forceps can be used to remove the cover slip. When using forceps to remove a cover slip, avoid contacting the hybridized surface directly as scratches can reduce the quality of the data. NOTE: Wash buffers 1, 2 or 3 for short oligonucleotide (<25-mer) microarrays.

16. Following a 5 min wash in Wash Buffer A, transfer the microarray slide to a wash station or beaker containing Wash Buffer B. For hybridizations involving probe and targets pairs of >100 bp, Wash Buffer B at 25°C for 5 min works well to remove the remaining un-hybridized material. After a 5 min incubation in Wash Buffer B, the microarray slide can be transferred to Wash Buffer C for 30 sec at 25°C. The microarray slide is then dried with a Microarray High-Speed Centrifuge and scanned.

Figure 3. Arrayit offers hybridization cassette in different colors for ease of use. Please see Ordering Information to purchase (1) AHC (black), (2) AHCA (amethyst), (3) AHCTZ (topaz), (4) AHCO (peridot), (5) AHCC (citrine), and (6) AHCR (ruby). All six cassettes accommodate standard 1 x 25 x 76 mm glass substrate slide microarrays.

Figure 4. Arrayit Hybridization Cassette Extra Deep AHCXD (right) shown next to a standard Hybridization Cassette AHC (left). The Extra Deep Hybridization Cassette has a 2.5 mm deep chamber (red arrow) instead of the standard 1.5 mm deep chamber, allowing the AHCXD and AHCPXD products to accommodate thicker substrates and slides, sandwich hybridizations with involving two 1.0 mm substrates, or a 1.0 mm substrate and a 1.0 mm thick cover slip.

Figure 5. Arrayit Hybridization Cassette Plus Extra Deep (Cat. AHCPXD) with custom 2.5 mm deep hybridization chambers to accommodate substrate slides thicker than 1.0 mm. AHCPXD cassettes can also be used to hybridize standard 1.0 mm substrate slides including Arrayit SuperEpoxy 3 Barcoded SME3BC (shown).

Recommended Equipment and Reagents
NanoPrint™ 2 Microarrayers
SpotBot® 4 Personal Microarrayers
InnoScan® Microarray Scanners
Array Plate Multi-Well Hybridization Stations
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
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