Corning® Epoxide Coated Slides - Instruction Manual
Contents
1. 40081 Corning 25 Slide Holders 10 20 To learn about Corning microplates and other laboratory products visit www corning com lifesciences 8 When using M Series LifterSlips place cover glass over array and carefully pipette Pronto Hybridization Solution so that it is drawn by capillary force into the space between the cover glass and the array Transfer array cover glass assembly to Corning Hybridization Chamber II Cat No 40080 only 3 Assemble the chamber as described in the Corning Microarray Hybridiza tion Chamber Operating Instructions Manual Keep the chambers right side up and in a horizontal position at all times to prevent movement of the cover glass relative to the array 4 Submerge chamber array assembly in a water bath or place in a hybridiza tion oven kept at 42 C 5 Hybridize arrays at 42 C for 12 to 16 hours Post Hybridization Washes It is extremely important not to allow the arrays to dry out between washes as this will result in high backgrounds Multiple containers are needed to perform the washes in the most efficient manner Have all containers and the volumes of washing solutions ready before starting the procedure Note that steps 2 and 3 both require solutions prewarmed to 42 C 1 Disassemble the hybridization chambers 2 Immerse arrays in 2 x SSC 0 1 SDS at 42 C until the cover glass moves freely away from the slide 3 Transfer arrays to 2 x SSC 0 1 SDS at 42 C for 52. Carefully lower the cover glass onto array Avoid trapping air bubbles between the array and the cover glass Small air bubbles that do form usually dissipate during hybridization Transfer array cover glass assembly to Corning Hybridization Chamber Cat Nos 2551 or 40080 6 2 Warm prehybridization solution to 42 C 3 Immerse arrays in prehybridization solution and incubate at 42 C for 45 to 60 minutes 4 Transfer prehybridized arrays to 0 1 x SSC and incubate at ambient temperature 22 to 25 C for 5 minutes 5 Repeat Step 4 twice for a total of three washes 6 Transfer arrays to purified water and incubate at ambient temperature for 30 seconds 7 Dry arrays by blowing high purity N2 over the array or by centrifugation at 1 600 x g for 2 minutes Keep arrays in a dust free environment while completing the preparation of the hybridization solution Preparation of Hybridization Solution The quality and purity of the template RNA and the resulting cDNA are critical factors for successful hybridizations Determine the yield and purity of the template RNA by measuring absorbance at 260 and 280 nm and by gel analysis Use only RNA showing a 260 280 ratio between 1 7 to 2 1 After synthesis and purification of the cyanine labeled target cDNA measure absorbance at 260 550 and 650 nm Best hybridization results are obtained with cDNA having a frequency of incorporation FOI of at least 20 labeled nucleotides per thousand
3. 9 Corning Microarray Products 9 3 OGT as to whether a license is necessary and if so secure one To inquire about a license under OGT s oligonucleotide array patents please contact licensing ogt co uk For information about OGT please visit its website at www ogt co uk 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 F O L I G O N U C L E OT I D E A R R AY S General Considerations Concentration of Printed Oligonucleotides The high reactivity of the Epoxide Coated Slides allows the use of dilute spotting solutions Optimal oligo nucleotide concentration for spotting on the Epoxide Coated Slides is between 20 and 50 M 50 M is approximately 0 5 mg mL for 30 mers When too little DNA is used the printed spots will not reach signal satura tion levels thus reducing the dynamic range of the array Conversely high ly concentrated printing solutions can produce spots with comet tails and other forms of localized background The concentration and purity of the DNA should be checked spectrophotometrically Desalted or HPLC puri fied oligonucleotides may be used Both amino modified and unmodified oligonucleotides form covalent bonds with the epoxide groups of the sur face of the slides Arrayer Settings and Pin Quality Follow the instructions provided by the manufacturer of arraying equipment and printing pins Pin contact time and the force with which t
4. T S Introduction 1 Overview 1 Handling and Care Instructions 1 Storage Instructions 2 Safety Considerations 2 Product Use Limitations Warranty Disclaimer 2 Preparation and Hybridization of Oligonucleotide Arrays 3 General Considerations 3 Array Fabrication and Stabilization 4 Array Hybridization 5 Prehybridization 5 Preparation of Hybridization Solution 6 Hybridization 7 Post Hybridization Washes 8 Additional Information 9 Customer Service and Technical Support
5. amount of cDNA containing 3 pmoles of each Cy3 and Cy5 dCTP in 12 L of hybridization solution For Cy cDNA made from total RNA use 1 0 pmoles of incorporated nucleo tides per microliter of hybridization solution per dye For example to hybridize an area covered by one Corning 22 x 22 mm cover glass approximately 5 cm2 dissolve an amount of cDNA containing 12 pmoles of each Cy3 and Cy5 dCTP in 12 L of hybridization solution 4 Dissolve the appropriate amount of target cDNA in the required volume of hybridization solution 5 Incubate the cDNA hybridization solution at 95 C for 5 minutes 6 Briefly centrifuge the cDNA hybridization solution to collect condensation and allow it cool to room temperature Do not place the solution on ice as this will cause precipitation of some of the components Protect the labeled cDNA from overexposure to light to minimize photobleaching Hybridization 1 Wash the required number of pieces of cover glass at least 1 piece of cover glass per array should be processed with nuclease free water followed by ethanol Dry cover glass by blowing high purity compressed N2 or allow to air dry in a dust free environment 2 Carefully pipette the target cDNA onto the arrayed surface Avoid touching the array with the pipette tip and creating air bubbles When using Corning Cover Glass Cat Nos 2870 22 2940 244 and 2940 246 apply the target cDNA in small volumes along the middle of the array
6. minutes 4 Transfer arrays to 1 x SSC at room temperature for 2 minutes 5 Repeat step 4 6 Transfer arrays to 0 1 x SSC at room temperature for 1 minute 7 Repeat Step 6 8 Dry arrays by blowing clean compressed N2 or by centrifugation at 1 600 x g for 2 minutes 9 Store arrays in a Corning 25 Slide Holders Cat No 40081 Protect arrays from overexposure to light until ready to scan Note Arrays fabricated on Epoxide Coated slides can be hybridized at tem peratures up to 65 C The use of hybridization temperatures higher than 42 C however calls for changes in the composition of the hybridization and wash solutions described in this manual such as exclusion of formamide or adjustment of salt concentrations to properly adjust their stringency to the requirements of the application at hand
7. Corning Incorporated Life Sciences 45 Nagog Park Acton MA 01720 U S A t 800 492 1110 t 978 635 2200 f 978 635 2476 www corning com lifesciences Worldwide Support Offices ASIA Australia t 61 2 9416 0492 f 61 2 9416 0493 China t 86 21 3222 4666 f 86 21 6288 1575 Hong Kong t 852 2807 2723 f 852 2807 2152 India t 91 11 341 3440 f 91 11 341 1520 Japan t 81 0 3 3586 1996 1997 f 81 0 3 3586 1291 1292 Korea t 82 2 796 9500 f 82 2 796 9300 Singapore t 65 6733 6511 f 65 6735 2913 Taiwan t 886 2 2716 0338 f 886 2 2716 0339 EUROPE France t 0800 916 882 f 0800 918 636 Germany t 0800 101 1153 f 0800 101 2427 The Netherlands amp All Other European Countries t 31 0 20 659 60 51 f 31 0 20 659 76 73 United Kingdom t 0800 376 8660 f 0800 279 1117 LATIN AMERICA Brasil t 55 11 3089 7419 f 55 11 3167 0700 Mexico t 52 81 8158 8400 f 52 81 8313 8589 2004 Corning Incorporated Printed in USA 6 04 CLS CS 022REV1 Corning is a registered trademark of Corning Incorporated Corning NY Discovering Beyond Imagination Flame of Discovery design and Pronto are trademarks of Corning Incorporated Corning NY Amersham Bioscience and Cy are registered trademarks of Amersham plc Buckinghamshire England FluoroLink is a trademark of Amersham Biosciences Limited or its subsidiaries Buckinghamshire England LifterSlip is a trademark of Apogent Portsmouth NH Corning Incorporated One Riverfront Pla
8. Using cDNA of lower FOI reduces the sensitivity of the assay An FOI greater than 50 is indicative of incomplete removal of unincorporated labeled nucleotides Determine the yield and label strength of target cDNA using the following formulae Amount of target cDNA ng A260 x 37 x total volume of cDNA L Labeled nucleotides incorporated pmoles for Cy 3 A550 X total volume of cDNA 0 15 for Cy 5 A650 X total volume of cDNA 0 25 FOI Labeled nucleotides incorporated x 324 5 amount of target cDNA Note These equations were generated using the following constants One A260 unit of single stranded DNA 37 g mL Extinction Coefficient of Cy3 150 000 M 1cm 1 at 550nm Extinction Coefficient of Cy5 250 000 M 1cm 1 at 650 nm Average Molar Mass of dNTP 324 5 1 Prepare fresh hybridization solution consisting of For short oligonucleotides 30 mers 10 formamide 5 x SSC 0 1 SDS and 0 1 mg mL of a nucleic acid blocker such as sonicated salmon sperm DNA or calf thymus DNA For long oligonucleotides 50 to 70 mers 20 to 35 formamide 5 x SSC 0 1 SDS and 0 1 mg mL of a nucleic acid blocker such as sonicated salmon sperm DNA or calf thymus DNA 9 A D D I T I O N A L I N F O R M AT I O N Customer Service and Technical Support For a detailed troubleshooting guide end user FAQ and additional product information please visit www corning com lifesciences For questions further clarification about this proto
9. aturated NH4Cl and KNO3 will provide 69 humidity at 30 C Saturated NH4Cl and KNO3 will provide 71 2 humidity at 25 C Saturated NH4Cl and KNO3 will provide 72 6 humidity at 20 C 4 Store arrays in original orange plastic container or in a 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 pre vent oxidation of spotted material and extends the shelf life of the arrays Array Hybridization Most microarray experiments are designed to measure relative transcript abundance transcriptional profiling for which it is necessary to convert RNA into fluorescently labeled cDNA This instruction manual describes labeling parameters and hybridization protocols related to this application Other applications for which DNA microarrays made on Epoxide Coated Slides are also used may involve the labeling of other types of nucleic acids such as genomic DNA and short oligonucleotides and the use of other enzymes such as DNA polymerases and terminal transferases For tran scriptional profiling we recommend the synthesis of cDNA by reverse transcription of total or mRNA in the presence of cyanine labeled dCTP We specifically recommend the use of the Pronto Plus Systems Cat Nos 40051 to 40056 for RNA isolation cDNA synthesis and array hybridization Preh
10. col and other technical issues and infor mation not covered in this manual please e mail clstechserv corning com or call 800 492 1110 1 978 635 2200 outside Canada and USA Corning Microarray Products Cat No Product Description Qty Pk Qty Cs 40041 Epoxide Coated Slides with Bar Code 5 25 40042 Epoxide Coated Slides without Bar Codea 5 25 40040 Epoxide Coated Slide Starter Kit with 5 mL Epoxide 5 10 Spotting Solution and 0 8 mL Hybridization Solution 40047 Pronto Epoxide Spotting Solution 250 mL 1 1 40028 Pronto Universal Hybridization Kit for 10 Arrays 1 1 40026 Pronto Universal Hybridization Kit for 25 Arrays 1 1 2551 Hybridization Chamber 1 5 40080 Hybridization Chamber II with Increased Depth 1 5 40001 Hybridization Chamber O rings 5 5 2870 22 Corning Cover Glass Square 22 x 22 mm 1 oz 10 packs No 11 2 2940 244 Corning Cover Glass Rectangular 24 x 40 mm 1 oz 10 packs No 11 2 2940 246 Corning Cover Glass Rectangular 24 x 60 mm 1 oz 10 packs No 11 2 3357 96 Well V bottom Polypropylene Microplate 25 100 3656 384 Well Polypropylene Storage Microplate 25 100 3672 384 Well Microarray Printing Plate Low Volume 10 50 3099 Universal Lid Rigid Lid for 96 and 384 Well 25 50 Microplates 6569 Aluminum Sealing Tape for 384 Well Blocks and 100 100 Microplates 6570 Aluminum Sealing Tape for 96 Well Blocks and 100 100 Microplates
11. ducts are guaranteed to perform as described when used properly Manufacturer liability is limited to the replacement of the product or a full refund Any misuse of this product including failure to follow proper use protocols is the responsibility of the user and Corning makes no warranty or guarantee under these circumstances Certain arrays and or methods of preparation analysis or use may be covered by intellectual property rights held by others in certain countries Use of this product is recommended only for applications for which the user has a license under proprietary rights of third parties or for technology for which a license is not required Corning s products may be used in connection with the manufacture use and or analysis of oligonucleotide arrays under patents owned by Oxford Gene Technology Limited or related companies OGT but Corning does not have the right to pass on a license 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 with 5 enclosed in an airtight container such as an acrylic desiccator or a glass desiccator jar A small glass dish can be used to hold the saturated salt solution in the bottom of the desiccator and humidity can be monitored with a hygrometer Recommended salt solutions are Saturated sodium nitrite NaNO2 will provide 66 humidity at 20 C S
12. ed in the Pronto Universal Hybridization Kits Array Fabrication and Stabilization The Pronto Epoxide Spotting Solution Cat No 40047 is provided ready for use Dilution or addition of other reagents is not necessary This spotting solution is an excellent medium for dissolving single stranded oligonucleo tides for printing microarrays This proprietary formulation has been tested thoroughly on Epoxide Coated Slides and may be used with either solid or quill pins Please note that it is not necessary to UV crosslink or bake the arrays to achieve covalent attachment of the oligonucleotides 1 Prepare DNA source plates sterile nuclease free Corning 384 well poly propylene microplates are recommended Cat Nos 3656 or 3672 by one of either alternative methods a or b Sufficient volume of printing solution needs to be prepared to cover the bottom of the receiving wells this cor responds to between 5 to 10 L per well when using 384 well microplates of standard well volume Please follow the recommendations of the microarrayer manufacturer a Dissolve oligonucleotides to a concentration of 20 to 50 M see General Considerations for details page 3 in the spotting solution Transfer DNA solution to a Corning 384 well microplate b Alternatively add the desired volume of spotting solution to wells containing DNA that has been dried by vacuum centrifugation 2 Set up arrayer and print slides according to the arrayer manufactur
13. er s or laboratory protocol The printing environment should be free of dust particles and kept at a temperature of 20 to 22 C with relative humidity between 55 and 70 3 Incubate printed arrays at 70 to 75 relative humidity i e in a humidity oven and at a temperature of 20 to 22 C for 12 to 17 hours The print ing instrument can also be used for this step if humidity can be controlled Alternatively create a humidity chamber by using a saturated salt solution 7 For double stranded cDNA 30 to 50 formamide 5 x SSC 0 1 SDS and 0 1 mg mL of a nucleic acid blocker such as sonicated salmon sperm DNA or calf thymus DNA 2 Determine the area of the slide to be exposed to the hybridization solution and calculate the volume of hybridization solution needed for each array When using Corning Cover Glass Cat Nos 2870 22 2940 244 and 2940 246 apply 2 5 L of hybridization solution per cm2 of surface area When using M Series LifterSlip apply 3 L per cm2 3 Calculate the amount of target cDNA needed for each array The fluorescence strength required to achieve high levels of sensitivity and broad dynamic range depends on the type of RNA used to synthesize the target cDNA For Cy cDNA made from mRNA use 0 25 pmoles of incorporated nucleo tides per microliter of hybridization solution per dye For example to hybridize an area covered by one Corning 22 x 22 mm cover glass approximately 5 cm2 dissolve an
14. fter removing slides to maintain a closed environment for unused slides Place the closed container in the pouch to protect the remaining slides and store them in a desiccator Use the remaining slides within one week of opening the pack Storage Instructions Store Epoxide Coated Slides at ambient temperature 20 to 25 C in origi nal undamaged packaging and use slides by the date indicated on the label Proceed as described in the Handling and Care Instructions after opening the package Safety Considerations When working with the Epoxide Coated Slides please follow all generally accepted laboratory safety guidelines At a minimum wear the appropriate personal protective equipment such as a lab coat safety glasses powder free latex gloves etc Follow recommended standard operating procedures for any laboratory equipment used in your experiments Read all Material Safety Data Sheets MSDS for appropriate handling of all reagents MSDS are available upon request or can be downloaded from www corning com lifesciences Product Use Limitations Warranty Disclaimer Corning Epoxide Coated Slides are sold for research purposes only and are not intended for resale This product is not to be used in human diagnostics or for drug purposes nor is it to be administered to humans in any way This product contains chemicals that may be harmful if misused Proper care should be exercised with this product to prevent human contact Corning pro
15. he pin strikes the slide affect spot size and mor phology Pins must be individually qualified before use Pins that are either broken or do not conform to specifications can ruin otherwise good arrays Make sure to optimize the printing and pin washing steps before using the Pronto Epoxide Spotting Solution Cat No 40047 for the first time Solid pins with pin diameter of 120 m give spot sizes in the range of 70 to 100 m depending upon pin material Care must be taken to thoroughly wash the pins between visits to source wells in order to avoid sample carry over For example effective washing of quill pins can be accomplished by performing 10 cycles each of a 2 second rinse in water and 1 second in vacuum alternatively solid pins can be washed in a sonicating water bath for 20 seconds followed by 8 seconds of drying Background Fluorescence The sensitivity specificity and reproducibility of microarray hybridization are negatively affected by background fluores cence Depending on their age the storage conditions and the purity of the biological material and other components of the spotting solution used DNA microarrays may develop high levels of background fluorescence on and around the printed areas decreasing the specificity of the hybridization signals The occurrence of spotted fluorescence can be minimized by 2 Open the pouch just prior to printing Close the cap on the slide container as soon as possible a
16. ned for mechanical defects and the presence of dust and glass particles The epoxide surface is applied in an environmentally controlled HEPA filtered ISO Class 5 facility resulting in coated slides with highly uniform surface properties and low autofluorescence Surface wettability is consistent across the slide surface to assure uniform spot size and shape and to avoid uncon trolled wicking or poor volume transfer during the print Packaging has been developed to maintain the appropriate storage environment Handling and Care Instructions To maximize the benefits of using Corning premium substrates please follow these recommendations Use the slides in a clean environment Particles falling onto the slide sur face may cause defects in the printed array as well as nuclease contamina tion Self contained printing environments may be required to prevent such contamination Avoid direct contact with the surface of the slide Only the print pins and processing solutions should touch the print area to avoid contamination and abrasion of the coating When using slides without bar codes clearly mark the side to be printed using a glass etching tool If the package of slides has been inadvertently stored at temperatures lower than 20 C allow it to come to ambient temperature 20 to 25 C before opening Otherwise condensation may form on the slide surface negatively affecting the uniformity of the coating C O N T E N
17. ybridization Prehybridization should be done immediately preceding the application of the target cDNA onto the arrays This step has the purpose of blocking the unused surface of the slide and removing loosely bound probe DNA It is recommended that all target cDNAs be characterized prior to the start of prehybridization The preparation of the hybridization solutions can be completed during the time arrays are being prehybridized 1 Prepare prehybridization solution consisting of 5 x SSC 0 1 SDS and 0 1 mg mL BSA The volumes required to process a given number of arrays depends on type of glassware available Use Coplin jars to simul taneously process up to 5 arrays using only 50 mL of solution per step 4 placing arrays in a Corning 25 Slide Holders Cat No 40081 and stor ing them in clean desiccators This form of background fluorescence can be eliminated by processing the arrays with the presoaking reagents includ ed in the Pronto Universal Hybridization Kits Cat Nos 40026 40028 The spurious attachment of labeled DNA to the unprinted area of the slide causes high background that interferes with spot identification during data collection and limits the sensitivity and dynamic range of the array Deactivating and or blocking the unused surface of the slide greatly reduces the incidence of this form of background and can be achieved by processing the arrays with the presoaking and prehybridization reagents conveniently includ
18. za Corning NY 14831 0001 Corning Epoxide Coated Slides Instruction Manual For Research Laboratory Use Cat No 40040 Epoxide Coated Slide Starter Kit Cat No 40041 Epoxide Coated Slides with Bar Code Cat No 40042 Epoxide Coated Slides without Bar Code For the most current information about these and related products please visit www corning com lifesciences Life Sciences I N T R O D U C T I O N Overview Corning Epoxide Coated Slides have a uniform coating of a proprietary epoxide chemistry that enables covalent attachment of unmodified and amino modified oligonucleotides to the glass substrate The coating is applied on both sides of the slides using a proprietary process under tightly controlled manufacturing conditions The slides offer a printing surface of unmatched cleanliness high DNA binding capacity uniformity and stability Microarray quality is highly dependent on the quality and integrity of the printing substrate Arrays printed on coated glass of poor quality are likely to produce spots of varying size shape and DNA content The presence of scratches haze and contaminating particulates on the slide surface also cause deformation of the arrays as well as high background fluorescence These problems lead to loss in sensitivity and generally poor results Epoxide Coated Slides are manufactured under the most stringent conditions to prevent these problems All slides are cleaned and individually exami
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