Pronto! Universal Microarray Kits

Corning’s products may be used in connection with the manufacture, useand/or analysis of oligonucleotide arrays under patents owned by OxfordGene Technology Limited or related companies (“OGT”), but Corning doesnot have the right to pass on a licence under any such patents. Therefore,before Corning’s products can be used in connection with the manufacture,use, or analysis of oligonucleotide arrays, the user should first check withOGT as to whether a licence is necessary and if so, secure one. To enquireabout a licence under OGT's oligonucleotide array patents, please contactlicensing@ogt.co.uk. For information about OGT, please visit its websiteat www.ogt.co.uk. Note: It is not necessary to UV crosslink or bake arrays printed on Epoxide Slidesto covalently attach DNA. RNA Integrity. The RNA concentration and purity should be determinedby the absorbance at 260/280 nm and gel analysis. The A260/280 ratioshould be 1.8 to 2.0. The purity and integrity of the RNA should beconfirmed by gel electrophoresis. To check for DNA contamination, analiquot of RNA may be digested with RNase and run on an agarose gel.The presence of a smear or bands after RNase treatment is indicative ofDNA contamination. DNA contamination will result in low signals andhigh background after hybridization. Input of Labeled cDNA. The optimal frequency of incorporation (FOI # of dye-labeled nucleotides per 1,000 nucleotides) is between 20 and 50dye-labeled nucleotides per 1,000 nucleotides. Lower incorporation willaffect the sensitivity of the labeled target. An FOI greater than 50 dyelabeled nucleotides per 1,000 is also sub-optimal due to low hybridizationefficiencies believed to be due to steric hindrance from the cyanine dyemolecules. Background Fluorescence. Depending on their age, the purity of the biologicalmaterial and other reagents used, and the storage conditions, DNA microarrays may develop significant levels of background fluorescence on andaround the printed areas. It is important to eliminate “spotted” fluorescencein order to accurately measure basal levels of transcript abundance. ThePronto! Universal Pre-Soak treatment followed by Pre-Hybridization,as detailed below, is very efficient at eliminating background fluorescence.P R E PA R AT I O N A N D H Y B R I D I Z AT I O N O FD N A M I C R O A R R AY SGeneral ConsiderationsThe surfaces of UltraGAPS and Epoxide Coated Slides are highly reactivetowards DNA. The key to producing microarrays of high quality is to takeadvantage of this high reactivity during the printing process while minimizing the spurious attachment of nucleic acids to the unprinted area duringsubsequent manipulation of the array. The following are some of the mostcritical factors to consider: 4Immobilization Procedures. UV cross-linking and/or baking enhances binding of DNA to the GAPS coated surface. These procedures work equallywell for DNA molecules longer than 300 bp. Smaller DNA molecules andoligonucleotides are best immobilized by UV cross-linking. When baking,care should be taken regarding the cleanliness of the oven. Volatile organicscan irreversibly contaminate the surface of the array leading to highbackgrounds.Concentration of the DNA. The high reactivity of UltraGAPS and EpoxideSlides allows the use of dilute printing solutions. The optimal concentration needs to be determined empirically. When too little DNA is used,the printed spots will not reach signal saturation levels, thus reducing thedynamic range of the array. Conversely, highly concentrated printingsolutions can produce spots with “comet tails” and other forms of localizedbackground. The concentration and purity of the DNA should be checkedspectrophotometrically as well as electrophoretically. We recommend0.1 mg/mL as a starting point for further optimization when printingdsDNA (e.g., PCR products, genomic DNA) and 0.5 mg/mL whenprinting oligonucleotides.Printing ProtocolArrayer Settings and Pin Quality. Follow the instructions provided by themanufacturer of arraying equipment and printing pins. Pin-contact timeand the force with which the pin strikes the slide affect spot size and morphology. Pins must be individually qualified before use. Pins that are eitherbroken or do not conform to operating specifications can ruin otherwisegood arrays. Make sure to optimize the printing and pin-washing stepsbefore using the Pronto! Universal Spotting Solution with UltraGAPSSlides or Pronto! Epoxide Spotting Solution with Epoxide Slides for thefirst time.The Pronto! Universal Spotting Solution (Cat. No. 40027) and Pronto!Epoxide Spotting Solution (Cat. No. 40047) are provided ready for use.Dilution or addition of other reagents is not necessary. The Universal SpottingSolution is an excellent medium for dissolving oligonucleotides as well asdsDNA for printing microarrays. This proprietary formulation has been testedthoroughly on UltraGAPS Slides and may be used with either solid or quillpins. Alternatively, the Pronto! Epoxide Spotting Solution is formulated foruse with Corning Epoxide Slides.5

For Oligonucleotide Arraysa. Dissolve dsDNA to a maximum of 0.25 mg/mL (0.1 mg/mL is a goodstarting concentration for further optimization) in Pronto! UniversalSpotting Solution if using UltraGAPS Slides or Pronto! EpoxideSpotting Solution if using Epoxide Slides. Transfer the DNA solutionto the Corning 384 well plate.1. Prepare DNA source plates (sterile, nuclease-free Corning 384-wellstorage microplates are recommended; Cat. No. 3656 and 3672) by one ofeither alternative methods a or b. A sufficient volume of printing solutionneeds to be prepared to cover the bottom of the receiving wells; this corresponds to between 5 and 10 µL per well when using 384 well plates ofstandard volume. Please follow the recommendations of the microarrayermanufacturer.b. Alternatively, add the desired volume of spotting solution to the wellscontaining DNA that have been dried by vacuum centrifugation.2. Set up the arrayer and print slides according to the manufacturer’s orlaboratory protocol. The printing environment should be free of dustparticles, and kept at a temperature of 20 to 22 C with relative humiditybetween 45 and 55%.a. Dissolve oligonucleotides to a maximum of 1.0 mg/mL (0.5 mg/mLis a good starting concentration for further optimization) in Pronto! Universal Spotting Solution if using UltraGAPS Slides or Pronto!Epoxide Spotting Solution if using Epoxide Slides. Transfer the DNAsolution to a Corning 384 well plate.3. Place arrays in a desiccator for up to 48 hours (vacuum desiccator worksbest).b. Alternatively, add the desired volume of spotting solution to the wellscontaining DNA that have been dried by vacuum centrifugation.4. Immobilize spotted DNA printed on UltraGAPS slides by applying 150 to300 mJ of UV light to the printed surface of the array, or by baking thearray at 80 C for 2 to 4 hours.2. Set up the arrayer and print slides according to the manufacturer’s orlaboratory protocol. The printing environment should be free of dustparticles, and kept at a temperature of 20 to 22 C.Note: It is not necessary to UV crosslink or bake arrays printed on Epoxide Slidesto covalently attach DNA.Note: Please refer to the UltraGAPS Coated Slides Instruction Manual orthe Epoxide Coated Slides Instruction Maual for detailed printing protocols.5. Store arrays in a dry environment at normal laboratory temperature (20 to 25 C). The orange plastic containers in which the slides were originallypackaged may be used for long-term storage of the arrays. Arrays can bestored for up to 6 months prior to hybridization. Exchanging the regularatmospheric air for clean nitrogen gas helps prevent oxidation of spottedmaterial and extends the shelf life of the arrays.3. Place arrays in a desiccator for up to 48 hours (vacuum desiccator works best).4. Immobilize spotted oligonucleotides printed on UltraGAPS Slides byapplying 600 mJ of UV energy to the printed surface.Note: It is not necessary to UV crosslink or bake arrays printed on Epoxide Slidesto covalently attach DNA.Labeling Protocol5. Store arrays in a dry environment at normal laboratory temperature (20 to 25 C). The orange plastic containers in which the slides were originallypackaged may be used for long-term storage of the arrays. Arrays can bestored for up to 6 months prior to hybridization. Exchanging the regularatmospheric air for clean nitrogen gas helps prevent oxidation of spottedmaterial and extends the shelf life of the arrays.The Pronto! Long Oligo/cDNA Hybridization Solution and Pronto! ShortOligo Hybridization Solution have been validated using Cy -labeled cDNAsynthesized by reverse transcription of mRNA and total RNA in the presenceof Cy-dCTP. We have consistently obtained good yields and incorporationrates with the FluoroLink Cy3- and Cy5-dCTPs (Amersham BiosciencesCat. Nos. PA53021 and PA55021, respectively) for fluorescently labeledcDNA synthesis. We, therefore, strongly recommend these as a source forfluorescently labeled dCTPs. In addition, these hybridization solutions mayalso be used to dissolve other types of fluorescently labeled nucleic acids formicroarray hybridization.For Double-stranded-DNA Arrays1. Prepare DNA source plates (sterile, nuclease-free Corning 384-well storagemicroplates are recommended; Cat. No. 3656 and 3672) by one of eitheralternative methods a or b. A sufficient volume of printing solution needs tobe prepared to cover the bottom of the receiving wells; this corresponds tobetween 5 and 10 µL per well when using 384 well plates of standard volume.Please follow the recommendations of the microarrayer manufacturer.6The quality and cleanliness of the starting RNA and the resulting cDNA arecritical factors for successful use of the arrays. It is recommended that RNAquality be thoroughly checked before attempting to synthesize cDNA and7

that the labeled cDNA be purified and quantified with a spectrophotometer.Exposure of solutions containing fluorescent nucleotides to light should beminimized to prevent photo bleaching.3. Immerse arrays in solution from Step 2 and incubate at 42 C for 20minutes. 20 minutes is required for presoak, but extending the timebeyond 20 minutes is NOT advisable and may reduce signal.For a complete and updated listing of RNA purification and direct-labelingsystems compatible with the Pronto! Universal Microarray Kits, please visitwww.corning.com/lifesciences/pronto.4. Transfer arrays to Wash Solution 2 and incubate at ambient temperaturefor 30 seconds.5. Transfer arrays to a fresh container of Wash Solution 2 for 30 seconds.Hybridization Protocol6. Transfer arrays to 42 C Universal Pre-Hybridization Solution (fromStep 1) and incubate for 15 minutes.Preparation of Wash Solutions7. Transfer arrays to a fresh container of Wash Solution 2 and incubate atambient temperature for 1 minute.The volumes of Universal Wash Reagents A and B provided in the Pronto!Universal Validation and Hybridization Kits are sufficient for processing10 or 25 arrays. We recommend preparing wash solutions all at one timeas described in order to control variation in the preparation. The followingvolumes for wash solution preparation are for 10 microarrays and shouldbe adjusted by multiplying by 2.5 for the Pronto! Universal HybridizationKit (25 arrays, Cat. No. 40026). Carefully follow the order of addition.8. Transfer arrays to Wash Solution 3 and incubate at ambient temperaturefor 30 seconds.9. Transfer arrays to a fresh container of Wash Solution 3 and incubate atambient temperature for 30 seconds.10. Dip arrays in nuclease-free water at ambient temperature (22 to 25 C), anddry by blowing high-purity nitrogen gas over the array or by centrifugation.Wash Solution 1deionized water (18 MegaOhm Milli-Q preferred)Universal Wash Reagent AUniversal Wash Reagent BNote: To dry arrays by centrifugation, centrifuge the arrays at 1,600 g for2 minutes. Use 50 mL conical tubes to hold individual arrays or slide holders tohold multiple arrays.447.5 mL50 mL2.5 mL11. Store arrays in a dust-free environment (or in slide holder) until readyfor use.Wash Solution 2deionized water (18 MegaOhm Milli-Q preferred)Universal Wash Reagent A1,425 mL75 mLNote: Store arrays in original plastic container or Corning 25 Slide Holder(Cat. No. 40081) in a dry environment at ambient temperature (20 to 25 C).Arrays can be stored for 6 months prior to hybridization. Exchanging the regular atmospheric air for clean nitrogen gas helps prevent oxidation of spottedmaterial and extends the shelf life of the arrays.Wash Solution 3Wash Solution 2deionized water (18 MegaOhm Milli-Q preferred)300 mL1,200 mLPre-Soak and Pre-HybridizationHybridizationNote: We recommend processing 5 arrays in a TPX staining jar (VWR International,Cat. No. 25460-907) which requires 100 mL of each type of solution used for eachstep in the following protocol. If you choose to use an alternative wash vessel, pleaseadjust your volumes accordingly.1. Wash the required number of pieces of Corning Cover Glass (Cat. No.2870-22, 2940-244, 2940-246; at least 1 piece of cover glass per arrayshould be processed) with nuclease-free water, followed by ethanol. Drycover glass by blowing high-purity compressed nitrogen gas or allow toair-dry in a dust-free environment.1. Heat required volumes of both Pronto! Universal Pre-Soak Solution andPronto! Universal Pre-Hybridization Solution to 42 C for at least30 minutes.2. Dry the appropriate amount of each dye-labeled cDNA (see table onpg. 10) using a speed-vacuum concentrator.2. Add 1 Sodium Borohydride Pre-Soak Tablet to 100 mL of 42 CUniversal Pre-Soak Solution. Allow the tablet to dissolve completely(approximately 5 minutes). Do not add Sodium Borohydride Pre-SoakTablet more than 15 minutes before use.8a. For cDNA or long oligo nucleotide arrays ( 50-mer), dissolve the dyelabeled cDNA in the required volume of Pronto! Long Oligo/cDNAHybridization Solution (see table on pg. 10).9

b. For short oligo nucleotide arrays ( 30-mer), dissolve the dye-labeledcDNA in the required volume of Pronto! Short Oligo HybridizationSolution (see table below).glass on the array. Avoid trapping air bubbles between the array and thecover glass. Small air bubbles that do form usually dissipate duringhybridization. Assemble the hybridization chamber as described in itspackage insert.Calculating the Volume of Hybridization Solution to Use6. Incubate the chamber-array assembly at 42 C for 12 to 16 hours using awater bath or a hybridization oven.The volume of Pronto! Long Oligo/cDNA Hybridization Solution orShort Oligo Hybridization Solution needed depends on the size of theprinted area and cover glass. For the Corning cover glass, use 2.5 to 3.5µL of hybridization solution per cm2 of surface area. This range of volumewill accommodate differences in humidity conditions and hybridizationtimes. The fluorescence strength required to achieve high levels ofsensitivity and broad dynamic range depends on the template used tosynthesize the Cy -cDNA.Note: Do not allow arrays to dry out during the hybridization process.Post-Hybridization WashesNote: We recommend processing 5 arrays in a TPX staining jar (VWR International,Cat. No. 25460-907), which requires 100 mL of each type of solution used for eachstep in the following protocol. If you choose to use an alternative wash vessel, pleaseadjust your volumes accordingly.Recommended Hybridization Solution Volumes and pmol of labeled cDNABased on Varying Coverslip SizesCoverslipSize22 22 mm24 40 mm24 60 mmSurfaceArea (cm2)Volume ofHybridizationSolutionAmount ofLabeled cDNAfrom Total RNA(per slide)Amount ofLabeled cDNAfrom mRNA(per slide)4.849.6014.412-17 µL24-33 µL36-50 µL12-17 pmol24-33 pmol36-50 pmol3-4 pmol6-8 pmol9-12 pmolDo not allow the arrays to dry out between washes, as this irreversiblyincreases background levels. Multiple containers are needed to perform thewashes in the most efficient manner. Have all containers and the volumes ofwashing solutions ready before starting the procedure.Note: Always wash arrays hybridized with different probes separately and withfreshly prepared wash solutions.1. Heat required volume of Wash Solution 1 to 42 C for at least 30 minutes(note that Steps 3 and 4 both require prewarmed solutions).*If doing a two-color hybridization, combine the recommended amount of both dye-labeled cDNAs.For example, for a 22 22 mm coverslip with a two-color hybridization using total RNA-derived cDNA,combine 12-17 pmol of Cy3-labeled and 12-17 pmol of Cy5-labeled cDNAs.2. Disassemble the hybridization chambers with the printed array sidefacing up.Calculating the Amount of cDNA to Use3. Immerse arrays in Wash Solution 1 at 42 C for 1 to 2 minutes until thecover glass falls from the slide.Total RNA: For Cy-labeled cDNA made from total RNA, dry down1.0 pmol of labeled cDNA per microliter of hybridization solution thatwill be used per dye.4. Transfer arrays to a fresh container of Wash Solution 1 at 42 C andincubate for 5 minutes.mRNA: For Cy-labeled cDNA made from mRNA, dry down 0.25 pmolof incorporated nucleotides per microliter of hybridization solution thatwill be used per dye.5. Transfer arrays to Wash Solution 2 at ambient temperature (22 to 25 C)and incubate for 2 minutes.6. Repeat step 5 in a fresh container of Wash Solution 2.3. Incubate the labeled cDNA solution at 95 C for 5 minutes, protectingsamples from light.7. Transfer arrays to Wash Solution 3 at ambient temperature and incubatefor 2 minutes.4. Centrifuge the labeled cDNA at 13,500 g for two minutes to collectcondensation. Do not place the solution on ice because this will causeprecipitation of some of the components.8. Transfer arrays to a fresh container of Wash Solution 3 at ambienttemperature and incubate for 2 minutes.9. Dry arrays by blowing clean compressed nitrogen gas or by centrifugation.5. Place array in Corning Hybridization Chamber (Cat. Nos. 2551 or40080). Pipet the labeled cDNA gently up and down and then transferonto the surface of the printed side of the slide. Carefully place the cover1010. Store arrays in original plastic container or Corning 25 Slide Holder(Cat. No. 40081) in a dry environment at ambient temperature (20 to25 C).11

TRO U B L E S H O OTI N G G U I D E A N D C U STOM E RS E R V I C E I N F O R M AT I O NC O R N I N G P R O D U C T S F O R M I C R O A R R AY SCat. No.Product DescriptionFor a detailed troubleshooting guide, end-user FAQ and additional productinformation, please visit 6400284002740047400154001640017Pronto! Universal Validation KitPronto! Universal Printing KitPronto! Universal Hybridization Kit – for 25 ArraysPronto! Universal Hybridization Kit – for 10 ArraysPronto! Universal Spotting Solution – 250 mLPronto! Epoxide Spotting Solution – 250 mLUltraGAPS Coated Slides, with Bar CodeUltraGAPS Coated Slides, without Bar CodeUltraGAPS Coated Slides, with Bar Code,Bulk PackUltraGAPS Coated Slides, without Bar Code,Bulk PackEpoxide Coated Slides with Bar CodeEpoxide Coated Slides without Bar CodeHybridization Chambe

Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001 Pronto! Universal Microarray Kits Instruction Manual For Research Laboratory Use Only Cat.No.40024: Pronto! Universal Validation Kit Cat.No.40025: Pronto! Universal Printing Kit Cat.No.40026: Pronto! Universal Hybridization Kit– for 25 Arrays Cat.No.40028: Pronto!