ArrayIt® SuperNylon Microarray Substrates represent a highly innovative membrane surface for researchers wishing to utilize the time-tested attributes of positively charged nylon membranes in a microarray format. SuperNylon can be used to attach DNA, protein, carbohydrate and any other molecule that has been shown to bind nylon in traditional filter assays. The high binding capacity of the 150 µm nylon layer is complimented by a polished atomically flat glass substrate for optimal performance compatibility with all standard glass substrate slide microarrayers and scanners.

Description
Positively charged membrane coated glass substrate 25 x 76 mm
Corner chamfer for side orientation
150 µm positively charged nylon layer
Binding capacity DNA = 5 µg per square millimeter
Binding capacity Protein = 2 µg per square millimeter
Reaction area 25 x 76 mm

SuperNylon is compatible with most all microarray scanners including the InnoScan Series Scanners,  SpotLight Microarray Scanner and the SpotWare Colorimetric scanner. The following detection methods can be used with this surface:

· Fluorescence
· Colorimetric
· Chemiluminescence
· Radioactivity

SuperNylon substrates are ready to use “right out of the box” without any additional processing or surface preparation. The surface is positvely charged to allow crosslinking of DNA to the surface.  This surface is fully compatible with a variety of manufacturing technologies including ArrayIt® contact printing. BlockIt Blocking Buffer should be used for low background and to prevent non-specific binding in complex assays and hybridization reactions. DNA microarrays wash steps can be performed using Wash Buffers A, B and C.

SuperNylon Usage Tips
Printing the Microarray:
Proteins should be spotted in Protein Printing Buffer. DNA should be spotted in Micro Spotting Plus. If the protein is already in a 1X PBS solution, add an equal volume of printing buffer to achieve a final protein concentration between 0.1 and 2.0 mg/ml. Lyophilized oligonucleotides can be printed at 1 to 50 µM final concentration with phenol red (0.001%) as the tracking dye to monitor the microarray printing quality. DNA crosslinking should be done with 1,200 J of UV.  Allow spots do dry before cross linking.

SuperNylon is a 150-micron thick layer of Nylon that has a high binding capacity. When using a Robotic Microarrayer such as the NanoPrint, SpotBot 2 or other microarrayer in conjunction with an ArrayIt Spotting Device, decrease Z-axis speeds to 2 cm/sec to avoid damaging the membrane. Because of the hydrophilic nature of this substrate, keep dwell times of micro spotting pins to the surface at a minimum.  Print at 50 to 80% humidity and ambient temperatures should be used. For best source sample integrity, use a system like the SpotBot or NanoPrint with source plate cooling.

Printed Microarray Storage
Printed microarrays should be incubated overnight at low humidity to establish the coupling reaction. Only dry DNA can crosslink to the surface. Arrays can be stored in a clean, dry cool place. Refrigeration can be used, but Arrayit printing buffers are designed to stabilize proteins and DNA for long-term storage at room temperature. Vacuum packing can also be used; make sure microarrays are dry prior to vacuum packing. Store printed microarrays unblocked and un-washed, performing blocking and washing steps just prior to incubation or hybridization.

SuperNylon Blocking & Washing
Prior to performing binding reactions, SuperNylon should be blocked with BlockIt buffer for 1 hour and then washed 3 times in 1X PBS for 5 minutes using a high throughput wash station. Do not let the surface dry out between blocking, binding and washing reactions. SuperNylon should only be allowed to dry after printing and prior to scanning only. Use of an ArrayIt high throughput wash station will ensure success.

SuperNylon Incubation and Detection Reactions
Binding reactions can be 1 hour to overnight. Experiments must not be allowed to dry
out. Fluorescent reactions running for long periods of time should be kept in the dark. Secondary antibody reactions should be run for up to 2 hours and typically not less than 30 minutes. Concentration of detection antibody needs to be optimized empirically by the user. Colorimetric and other enzymatic detection reactions require optimization by the user. Biotin-labeled cRNAs, known as labeled cRNA targets, can be generated using TrueLabeling-AMP Linear RNA Amplification Kit following manufacturer’s protocol (SuperArray Bioscience Corporation). Briefly, total RNA (3 µg/array) can be converted to cDNA at 42°C for 50 minutes. These cDNAs are then in vitro transcribed to cRNAs in the presence of biotin-16-UTP (Roche Molecular Biochemicals, Basel, Switzerland, http://www.roche.com). Biotin-labeled cRNAs can be purified using a RNeasy Mini Kit (Qiagen). The concentration of cRNAs can be measured with a UV spectrophotometer (Amersham, Piscataway, NJ, http://www.amersham.com). Microarrays can be hybridized with biotin-labeled targets (5 µg/array) at 60°C for 17 hours. Filters can be washed with Wash buffes A, B and C at 60°C for 15 minutes each. Chemiluminescent detection steps can be performed by subsequent incubation of the microarrays with alkaline phosphatase–conjugated streptavidin and CDP-Star substrate.

Scanning
SuperNylon substrates are compatible with a variety of detection methods. The microarray should not be detected wet or damp unless the detection method is chemiluminescent. Air-dry the slide in a dark, dust free place until the membrane appears white. When imaging SuperNylon and other white surfaces in laser/pmt microarray scanners, the default laser/PMT for glass transparent surfaces are not optimal for best signal to noise ratio. Because of the increased binding capacity of the nylon 3-D layer lower laser power and PMT settings should be used. For best signal to noise rations in fluorescent detection use the SpotLight Microarray Scanner.

Scientific Publications
Click here for recent scientific publications using ArrayIt® brand SuperNylon Microarray Substrates from Arrayit International, Inc.

Recommended Equipment and Reagents
NanoPrint™ 2 Microarrayers
SpotBot® 4 Personal Microarrayers
InnoScan® Microarray Scanners
SpotLight™ 2 Microarray Scanners
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