Corning® Epoxide Coated Slides Instruction Manual
Contents
1. and when sub strate stability and consistency are absolute requirements Epoxide Coated Slides have a uniform coating of a proprietary epoxide chemistry that enables covalent attachment of unmodi fied and amino modified oligonucleotides to the glass substrate The coating is applied on both sides of the slides using a pro prietary process under tightly controlled manufacturing condi tions The slides offer a printing surface of unmatched cleanli ness 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 fluores cence These problems lead to loss of 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 examined 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 sha2. LATIN AMERICA Brasil t 55 11 3089 7419 f 55 11 3167 0700 Mexico t 52 81 8158 8400 f 52 81 8313 8589 CLS CS 022REV2 3 08 2008 Corning Incorporated Printed in USA
3. 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 OGT as to whether a license is necessary and if so secure one To inquire about a license under OGT oligonucleotide array patents please contact licensing ogt co uk For information about OGT please visit its website at www ogt co uk PREPARATION AND HYBRIDIZATION OF OLIGONUCLEOTIDE ARRAYS General Considerations Concentration of Probe Oligonucleotides The high reactivity of the Epoxide Coated Slides allows the use of dilute spotting solutions Optimal oligonucleotide concentration for spot ting on the Epoxide Coated Slides is between 20 and 50 pM 50 pM is approximately 0 5 mg mL for 30 mers When too little
4. mg mL BSA The volumes required to process a given number of arrays depends on type of glass ware available Use Coplin jars to simultaneously process up to 5 arrays using only 50 mL of solution per step 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 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 N 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 Deter mine 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 syn thesis and purification of the cyanine labeled target cDNA measure absorbance at 260 550 and 650 nm Best hybridiza tion results are obtained with cDNA having a frequency of incorporation FOI of at least 20 labeled nucleotides per thousand Using cDNA of lower FOI reduces the sensitivity of the assay An FOI greater than 5
5. 0 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 iL Labeled nucleotides incorporated pmoles for Cy 3 A s X total volume of cDNA 0 15 for Cy5 Asso 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 Az6 unit of single stranded DNA 37 pg mL Extinction Coefficient of Cy3 150 000 Mem at 550nm Extinction Coefficient of Cy5 250 000 M cm 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 2 Determine the area of the slide to be exposed to the hybridi zation 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 pL of hybridization solution per cm of surface area When using raised edge coverslips apply 3 pL per
6. 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 CORNING Corning Incorporated Life Sciences Tower 2 4th Floor goo Chelmsford St Lowell MA 01851 t 800 492 1110 t 978 442 2200 f 978 442 2476 www corning com lifesciences Corning is a registered trademark of Corning Incorporated Corning NY Worldwide Support Offices ASIA PACIFIC 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 91 124 235 7850 f 91 124 401 0207 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 6861 2913 Taiwan t 886 2 2716 0338 f 886 2 2716 0339 Pronto is a trademark of Corning Incorporated Corning NY All other trademarks in this document are the property of their respective owners Corning Incorporated One Riverfront Plaza Corning NY 14831 0001 EUROPE France t 0800 916 882 f 0800 918 636 Germany t 0800 101 1153 f 0800 101 2427 The Netherlands t 3120 655 79 28 f 3120 659 76 73 United Kingdom t 0800 376 8660 f 0800 279 1117 All Other European Countries t31 0 20 659 60 51 f 31 0 20 659 76 73
7. 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 CORNING CONTENTS IntrodUction 06sec aoe bad eee tee id kaa eee 1 Overview Aea bebe 48 ohh eee nas 1 Handling and Care Instructions 2 Storage Instructions sce anneren eaa ence ae ge Bie 2 Safety Considerations 0 00000 2 Product Use Limitations Warranty Disclaimer 3 Preparation and Hybridization of Oligonucleotide Arrays 000000 000005 4 General Considerations arricieicepri ninsis amsa 4 Array Fabrication and Stabilization 5 Array Hybridization 0 cee tse a ee nenga 7 Pre Hybridization 0 0000 cece 7 Preparation of Hybridization Solution 8 Hybridization esses ccesn aca ides ate ete doe gnte gala 10 Post Hybridization Washes 11 Additional Information 0005 12 Customer Service and Technical Support 12 Corning Microarray Products 13 INTRODUCTION Overview Corning Epoxide Coated Slides are recommended for the fabrication of oligonucleotide arrays for applications including transcriptional profiling and SNP detection
8. DNA is used the printed spots will not reach signal saturation levels thus reducing the dynamic range of the array Conversely highly concentrated printing solutions can produce spots with comet tails and other forms of local ized background The concentration and purity of the DNA should be checked spectrophotometrically Desalted or HPLC purified oligonucleotides may be used Both amino modified and unmodified oligonucleotides form covalent bonds with the epoxide groups of the surface of the slides Arrayer Settings and Pin Quality Follow the instructions pro vided by the manufacturer of arraying equipment and print ing pins Pin contact time and the force with which the pin strikes the slide affect spot size and morphology Pins must be individually qualified before use Pins that are either bro ken or do not conform to specifications can ruin otherwise good arrays Care must be taken to thoroughly wash the pins between visits to source wells in order to avoid sample carry over Sonication of the pins for 30 minutes prior to the start of the print run and in the case of long runs at one or more points within the run help keep the pins in good working order Background Fluorescence The sensitivity specificity and reproducibility of microarray hybridization are negatively affected by background fluorescence Depending on their age the storage conditions and the purity of the biological material and other components of the s
9. ambers 2 Immerse arrays in 2 x SSC 0 1 SDS at 42 C until the cover glass moves freely away from the slide Transfer arrays to 2 x SSC 0 1 SDS at 42 C for 5 minutes Transfer arrays to 1 x SSC at room temperature for 2 minutes Repeat step 4 Aun Bw Transfer arrays to 0 1 x SSC at room temperature for 1 minute Repeat Step 6 o N Dry arrays by blowing clean compressed N or by centri fugation 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 temperatures up to 65 C The use of hybri dization 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 for mamide or adjustment of salt concentrations to properly adjust their stringency to the requirements of the application at hand 12 ADDITIONAL INFORMATION 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 protocol and other technical issues and information not cov ered in this manual please e mail clstechserv corning com or call 800 492 1110 1 978 442 2200 outside Canada and USA Corning Micr
10. 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 nucleotides per microliter of hybridization solution per dye For example to hybridize an area covered by one Corning 22 x 22 mm cover glass approx imately 5 cm dissolve an amount of cDNA containing 3 pmoles of each Cy3 and Cy5 dCTP in 12 pL of hybridization solution 10 For Cy cDNA made from total RNA use 1 0 pmoles of incorporated nucleotides per microliter of hybridization solution per dye For example to hybridize an area cov ered by one Corning 22 x 22 mm cover glass approxi mately 5 cm dissolve an amount of cDNA containing 12 pmoles of each Cy3 and Cy5 dCTP in 12 pL 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 to cool to room tempera ture Do not place the solution on ice as this will cause pre cipitation of some of the components Protect the labeled cDNA from overexposure to light to minimize photo bleaching Hybridization 1 Wash the required number of pieces of cover g
11. ed Slides at ambient temperature 20 to 25 C in original 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 mini mum 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 purpos es 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 products 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
12. egular atmospheric air for clean nitrogen gas helps prevent oxidation of spotted material and extends the shelf life of the arrays Array Hybridization This instruction manual describes labeling parameters and hybridization protocols for measuring relative transcript abun dance transcriptional profiling which typically involves the synthesis of Cy cDNA by reverse transcription of total RNA or mRNA 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 transcriptional profiling we recommend the use of the Pronto Plus Systems Cat Nos 40055 and 40056 for direct labeling and 40075 and 40076 for indirect labeling which include reagents for RNA isolation cDNA synthesis and array hybridization Pre Hybridization 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 prehy bridization 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
13. ing solution to wells containing DNA that has been dried by vacuum centrifugation 2 Set up arrayer and print slides according to the arrayer man ufacturer 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 Optional Incubate printed arrays at 70 to 75 relative humidity i e in a humidity chamber kept at ambient tem perature 20 to 25 C for 12 to 17 hours The printing instrument can also be used for this step if humidity can be controlled Alternatively create a humidity chamber by using a saturated salt solution enclosed in an airtight con tainer such as an acrylic or 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 NaNO will provide 66 humidity at 20 C Saturated NH Cl and KNO will provide 69 humidity at 30 C Saturated NH Cl and KNO will provide 71 2 humidity at 25 C Saturated NH Cl and KNO will provide 72 6 humidity at 20 C 4 Place arrays in Corning 25 Slide Holder Cat No 40081 Place holder containing arrays in Corning Microarray Storage Pouch Cat No 40086 heat seal pouch and store in at ambient temperature Hybridize arrays within 6 months of fabrication Exchanging the r
14. lass with nuclease free water followed by ethanol Dry cover glass by blowing high purity compressed N j 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 Apply the target cDNA in small volumes along the middle of the array 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 II Cat No 40080 3 Assemble the chamber as described in the Corning Microarray Hybridization 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 hybridization 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 Multi ple containers are needed to perform the washes in the most efficient manner Have all containers and the volumes of wash ing solutions ready before starting the procedure Note that steps 2 and 3 both require solutions prewarmed to 42 C 1 Disassemble the hybridization ch
15. nce probe DNA is dissolved in a spotting medium it is very difficult to recover it for reconstitution in a different solvent The Pronto Epoxide Spotting Solution Cat No 40047 is provided ready for use Dilution of the Epoxide Spotting Solution or addition of other reagents is not necessary Customers are encouraged to try their own phosphate buffered spotting media in order to determine which medium produces the best results Sodium phosphate at a concentra tion of 150 mM pH 8 5 has been used very successfully on the Corning Epoxide Coated Slides Solutions containing DMSO do not work well on epoxide slides 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 polypropylene 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 pre pared to cover the bottom of the receiving wells this cor responds to between 5 to 10 pL 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 pM 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 spott
16. oarray Products Cat No Product Description Qty Pk Qty Cs 40041 Epoxide Coated Slides with Bar Code 5 25 40042 Epoxide Coated Slides without Bar Code 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 40029 Pronto Background Reduction Kit for 50 Arrays 1 1 40055 Pronto Plus Direct System for 25 Reactions 1 1 with RNA Isolation 40056 Pronto Plus Direct System for 25 Reactions 1 1 without RNA Isolation 40075 Pronto Plus Indirect System for 25 Reactions 1 1 with RNA Isolation 40076 Pronto Plus Indirect System for 25 Reactions 1 1 without RNA Isolation 40080 Hybridization Chamber II 1 5 40081 Corning 25 Slide Mailer 20 20 40082 Corning 5 Slide Mailer 50 50 40085 Microarray Storage Pouch for 5 Slides 100 100 40086 Miroarray Storage Pouch for 25 Slides 100 100 2870 22 Corning Cover Glass Square 22 x 22 mm No 1 2 1oz 10 packs 2940 244 Corning Cover Glass Rectangular 24 x 40 mm loz 10 packs No 11 2 2940 246 Corning Cover Glass Rectangular 24 x 60 mm loz 10 packs No 12 3357 96 Well V bottom Polypropylene Microplate 25 100 3656 384 Well Polypropylene Storage Microplate 25
17. pe and to avoid uncontrolled 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 sub strates please follow these recommendations Use the slides in a clean environment Particles falling onto the slide surface may cause defects in the printed array as well as nuclease contamination Self contained printing envi ronments 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 con densation may form on the slide surface negatively affecting the uniformity of the coating Open the pouch just prior to printing Close the cap on the slide container as soon as possible after 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 Coat
18. potting 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 placing arrays in a Corning 25 Slide Holders Cat No 40081 and storing them in a Microarray Storage Pouch Cat No 40086 This form of background fluorescence can be eliminated by pro cessing the arrays with the presoaking reagents included 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 included in the Pronto Universal Hybridization Kits Array Fabrication and Stabilization It is crucially important to fully evaluate the performance of a particular spotting medium under conditions that are as close to working conditions as possible before committing large sets of probes to the formulation Thorough and properly controlled print tests must be done in order to ensure that the desired spot density and array uniformity is achievable O
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