Oligo GEArray User Manual
Contents
1. quality cooled CCD camera system No more than 8 GEArrays Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 16 Se should be photographed at one time in a 1 4 mega pixel image field to allow sufficient resolution for accurate digital image analysis of the GEArrays It is not necessary to remove the arrays from the HybPlate for CCD camera photography OR 2 Remove the GEArray from the hyb chamber blot off the excess CDP Star by touching a corner of the array to a clean absorbent tissue and place it in an imaging sheet to scan or expose to X ray film As many GEArrays as the user is comfortable handling and the physical size of the X ray file or scanner can accommodate may be imaged at the same time CDP Star is a registered trademark of Applera Corporation or its subsidiaries in the US and certain other countries D Image and Data Acquisition and Analysis 1 Image Acquisition NOTE Hold the membrane as shown on the left of Figure 2B with the bar code facing you on your right This side of the array should face your film or CCD camera in this orientation a Capture Medium After the CDP Star incubation the GEArray is now ready for image acquisition The chemiluminescent array image can be captured by a number of means Imaging stations with a high quality cooled CCD camera work the best Their advantages include lower image back2. B Total RNA Isolation Kit Recommendations See the RNA Preparation section of the Protocol for details For cultured cells ArrayGrade Total RNA Isolation Kit SuperArray GA 013 For animal tissues TRIzol from Invitrogen Catalog 15596 026 followed by the ArrayGrade Total RNA Isolation Kit GA 013 C Biotin UTP Required from another manufacturer for labeled target synthesis See the TrueLabeling AMP 2 0 Kit User Manual for more details D Stock Buffers 1 20X SSC Dissolve 175 3 g NaCl and 88 2 g sodium citrate dihydrate into 900 ml ddH20 Adjust pH to 7 0 with 1M HCI Dilute to 1 L with ddH20 Store at room temperature 2 20 SDS Dissolve 200 g sodium dodecyl sulfate in 1 L ddH20 Heat to 65 C if necessary to dissolve Store at room temperature E Laboratory Equipment GEArray Express Thermoshaker Catalog Number GXE TS2 for 16 arrays 2 HybPlate model or GXE TS4 for 32 arrays 4 HybPlate model GEArray Multi Chamber HybPlates and seals GA 035 Thermal cycler or several incubators CCD Camera Imaging Station Alpha Innotech Syngene Kodak UVP etc or alternative high sensitivity chemiluminescent detection system e g X ray film Water bath or constant temperature oven at 60 C RNase free tubes and pipets F Image and Data Analysis Software GEArray Expression Analysis Suite GA 021 for 3 months GA 022 for one year The GEASuite is a web based application run from a different address than our compa
3. Panel A displays an Agilent BioAnalyzer electropherogram of a high quality total RNA preparation showing sharp peaks without shoulders especially to the left of each peak for the 18S and 28S ribosomal RNA left to right Panel B right hand lane displays an analysis of the same high quality total RNA preparation by agarose gel electrophoresis demonstrating sharp bands especially at the bottom of each band for the 28S and 18S ribosomal RNA top to bottom Because some contaminants are difficult to detect by simply looking at RNA integrity and can be missed by UV spectrophotometry it is essential to choose the proper RNA isolation method for your biological sample as described above Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 10 Ss 3 Amount Considerations GEArray Express combined with its companion labeling method TrueLabeling AMP 2 0 may be used at maximum speed and convenience when at least 1 0 ug of total RNA is available at a concentration greater than 0 2 mg ml for every sample Under these conditions the labeling method produces enough labeled cRNA target in 3 hours for the GEArray Express analysis approximately 2 0 ug Alternatively when only as little as 100 ng of total RNA is available the TrueLabeling AMP 2 0 protocol may be modified to include an overnight step in order to increase the yield to a level sufficient to
4. Suite is designed to help easily and rapidly interpret the microarray results GEArray Express Benefits Pathway Focused Design The content includes the most relevant pathway or disease specific genes Reliable and Reproducible Minimizes sample to sample and day to day variation Speed and Simplicity Easy to handle one day protocol processes up to 32 arrays in one day Sensitive Requires as little as 100 ng total RNA and detects over 3 logs linear dynamic range Cost Effective Priced from 58 33 per array Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 ce Figure 1 Overview of the GEArray Express System procedure Gene Expression Profiling with Oligo GEArrays mr AAAA RNA TTETITTI EERE RERTE LEELLE E ones and Labeling 3 hours Within 1 Day a hours Chemiluminescent Detection Detection CCD camera of X ray film 1 1 2 hours Data Extraction and Analysis with GEArray Expression Analysis Sulte Technical Support 888 503 3187 301 682 9200 support superarray net www sSuperArray com GEArray Express User Manual Version 1 0 8 5 2005 BG Figure 2 Appearance of the GEArray Express Microarrays The dry nylon membrane arrays for GEArray Express are shipped and stored in an ExpressPak box The printed array spots in rows of 8 12 or 16 are initially visible as red dot
5. is not important washing and detection steps 1 Aspiration can be performed using any standard laboratory aspiration system such as a Pasteur pipet attached to a vacuum pump and liquid trap or a water vacuum system or even a serological pipet and Pipet Aid During the aspiration of buffers the tip of the pipet should be placed into one corner of the HybPlate chamber It is important to use low level vacuum to minimize the risk of the tip of the pipet grabbing the microarray membrane which can created artifacts on the final microarray image Avoid touching the aspirator tip to the probe spotted region of the GE Array 2 Pouring off buffers should be done so that the buffer flows slowly out of one of the HybPlate corners and care should be taken to avoid pouring the array off with the buffer After the HybPlate is empty it is then inverted and gently tapped on 2 or 3 paper towels to absorb any residual drops left on the hybridization chamber walls The surface tension of the buffer keeps the GEArray stuck to the bottom of the hybridization chamber d Procedural timing and mid protocol break points i The GEArray Express System is designed to offer either 1 day processing or it can be broken down into two major operations CRNA preparation and GEArray hybridization which can be performed on different days at the user s convenience However once the Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www
6. to insure good target synthesis and good GEArray results 3 Improper washing in 0 1X SSC solution Excessive washing of the membrane in the high stringency buffer Wash Solution 2 will strip off the hybridized probes Decrease the washing time 4 Improper hybridization temperature Check the actual temperature inside your hybridization oven with a thermometer The digital temperature reading on your hybridization oven could be several degrees off calibration As the temperature goes higher than 60 C the amount of specifically hybridized target will decrease until all targets and probes are denatured 5 Underexposure Try multiple exposures for various longer times B HIGH BACKGROUND Other than RNA quality the following factors may cause high background 1 Pre hybridization step incomplete Needs at least 30 minutes 2 Inaccurate AP streptavidin dilution Since the AP streptavidin is dissolved in a glycerol containing solution special caution should be taken when pipeting the small volume of AP streptavidin We suggest diluting no less than 2 ul of AP streptavidin into 16 ml of Buffer Q a 1 8 000 dilution Draw up the needed volume and carefully Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 19 s wipe the outside of the pipet tip with a laboratory wipe before dispensing the volume The final working AP streptavidin dilution can a
7. using NOTE CDP Star stock solution is very susceptible to degradation by bacteria contamination Use sterile pipettor tips when dispensing Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 12 Se entire Seal against the HybPlate This should be done before placing the plastic cover on the HybPlate speeeseeoeowersov EMM UMM 8 Se ee ees pea Sees ee ees bt bv TEMG eh ES er IMM GN Figure 4 Proper positioning 4 Oligo GEArrays in the GEArray Multi Chamber HybPlate for CCD camera photography of the microarrays Panel A displays one example of compactly positioned GEArrays that will allow optimal photographic framing Panel B shows a configuration that would create a large empty space pixels that are not displaying microarray probe intensities within the image when framed to photograph all the arrays in one image iii Multi Chamber HybPlate buffer removal techniques Two different methods are employed to remove solutions from the hybridization chambers in the HybPlate Aspiration is used when it is important to keep the solution from contaminating adjacent chambers hybridization or to keep the edges of the HybPlate dry for good sealing to the Multi Chamber Seal wetting and prehybridization Pouring is used for quick easy removal of buffers when cross chamber contamination
8. 66651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 Be CONTENTS I Introduction 4 ll Materials Provided l Ill Additional Materials Required T IV GEArray Express Process 8 A RNA Preparation and Quality Control 8 B TrueLabeling AMP 2 0 cRNA Synthesis and Labeling 10 C GEArray Express Hybridization Protocol 10 D Image and Data Acquisition and Expression Analysis 15 V Troubleshooting Guide 18 LIMITED PRODUCT WARRANTY FOR GEARRAY PRODUCTS This product is intended for research purposes only and is not intended for diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to a physical manufacturing defect but not due to an inability to validate by an independent method any results obtained using this product SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product Warranties are subject to change at any time without notice NOTICE TO PURCHASER THE PURCHASE OF GEARRAY PRODUCTS INCLUDES A LIMITED NONEXCLUSIVE LICENSE TO USE THE MEMBRANES FOR RESEARCH USE ONL
9. J SuperArray Bioscience Corporation User Manual Part 1018A BIOMOLGmbH Waidmannstr 35 22769 Hamburg info biomol de www biomol de Phone 49 40 8532600 or Fax 1 49 40 85326022 or biomol 0800 2466651 D 0800 2466652 D ee GEArray Express System THE HIGH THROUGHPUT PATHWAY SPECIFIC GENE EXPRESSION PROFILING SYSTEM see Purchaser Notification for limited use license and warranty information page 3 GEArray Express User Manual Version 1 0 8 5 2005 Bo S SuperArray Bioscience Corporation GEArray Express Catalog Numbers EHS EMM and ERN The High Throughput Pathway Specific Gene Expression Profiling System USER MANUAL ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 0800 2466651 D 49 40 8532600 international e FAX 0800 2466652 D 49 40 853260 22 international e ON LINE ORDER www biomol de e E MAIL info biomol de to place an order ts biomol de for technical support You may place orders by phone fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information MasterCard Shipping address Billing address For more information visit us at http www superarray com or http www biomol de SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21 704 USA Technical Support 0800 24
10. Mix 0 5 ml 20X SSC 2 5 ml 20 SDS and 97 ml ddH2O This should be split to two separate containers as it will be needed at 2 different temperatures 60 C and 37 C iii These Wash Solutions may be stored at room temperature for up to six months b 1x Buffer F dilution i 5X Buffer F may cloud during storage at 4 C so warm the solution to 37 C and invert the bottles several times to allow any precipitate to completely dissolve li Dilute 5X Buffer F with ddH20O five fold to prepare enough 1X Buffer F for at least one Plate You need 8 ml of 1X Buffer F for washing each GEArray therefore at least 70 ml should be prepared c Warming buffers i GEAhyb Wash Solution 1 and one container of Wash Solution 2 should stored at room temperature but should be placed in a 60 C water bath or oven well ahead of starting the hybridization protocol Invert the containers several times to promote complete dissolution of the buffer components These solutions must be at 60 C when applied the GEArray for consistent processing li Buffer Q may cloud during storage at 4 C so warm the solution to 37 C and invert the bottles several times to allow any precipitate to completely dissolve Remove this buffer from 4 C at least an hour before use to insure that it has time to cool to room temperature before being applied to the GEArray iii CDP Star is stored in the dark at 4 C and should be allowed to warm to room temperature for at least 30 minutes before
11. SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 15 Se Keeping the wash conditions and times consistent among all arrays being processed at the same time Is essential for consistent array image quality and results i Add 4 ml pre warmed Wash Solution 1 to each GEArray and aspirate the buffer from wells then invert and tap the HybPlate on paper towels Do not shake during this step but keep the HybPlate on the 60 C platform li Add 2 ml pre warmed Wash Solution 1 and incubate for 5 shaking at 60 C then pour off the buffer and tap the HybPlate on paper towels iii Add 2 ml pre warmed Wash Solution 2 and incubate for 2 shaking at 60 C Wash Solution 2 is a high stringency wash Excessive washing of the membrane with Wash Solution 2 will result in a loss of probe signal intensity 4 Detection Phase The following steps utilize the Chemiluminescent Detection Kit D 01 a HybPlate cooling i Leaving the HybPlate shaking in the GEArray Express Thermoshaker turn the temperature setting down to 37 Open the thermoshaker lid and keep it open until the unit cools to 37 during one of the steps below at which time it should be closed again Proceed directly to the next steps which are part of the cooling process li Pour off the Wash Solution 2 and tap the HybPlate on paper towels iii Add 2 ml room temperature Wash Solution 2 to every chamber in the HybPlate and incubate for 10 with shaking Even when hybridizing few
12. Y THIS LICENSE DOES NOT GRANT RIGHTS TO USE THE MEMBRANES FOR REPRODUCTION OF GEARRAY FILTERS TO MODIFY GEARRAY FILTERS FOR RESALE OR TO USE GEARRAY TO MANUFACTURE COMMERCIAL PRODUCTS WITHOUT WRITTEN APPROVAL OF SUPERARRAY BIOSCIENCE CORPORATION NO OTHER LICENSE EXPRESSED IMPLIED OR BY ESTOPPEL IS GRANTED U S PATENTS MAY COVER CERTAIN ISOLATED DNA SEQUENCES INCLUDED ON THE GEARRAY PRESENTLY IT IS NOT CLEAR UNDER U S LAWS WHETHER COMMERCIAL USERS MUST OBTAIN LICENSES FROM THE OWNERS OF THE RIGHTS TO THESE U S PATENTS BEFORE USING GEARRAY Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 Bo I INTRODUCTION The advancement of nucleic acid array technology has made it possible to analyze the expression of multiple genes in a single experiment However most commercial gene expression array products designed to include as many genes as possible produce a vast amount of data that is overwhelming and difficult to interpret In addition the cost of detection equipment and bioinformatics software is often prohibitive Based on SuperArray s cDNA GEArray Q and S Series microarrays our popular second generation of pathway specific gene expression arrays our Oligo GEArrays allow researchers to characterize gene expression associated with a specific biological pathway in a more comprehensive and cost effective manner All of the 60 mer oligonucl
13. ackground Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 17 Se c Saving your microarrays After obtaining your exposures allow the damp membranes to completely dry in the HybPlate or imaging sheet and store at room temperature If you find that you need to obtain additional exposures at a later date re wet the membrane with water and then repeat the Buffer G rinse and CDP Star incubation from the above protocol New exposures can then be obtained although they will have a slightly higher non specific background 2 Image Analysis and Data Acquisition amp Analysis No matter which medium you choose to capture the image save all images as electronic files in a grayscale 8 or 16 bit TIFF or alternatively in JPEG file format CCD Cameras and phosphor imagers usually acquire their images in this format or a proprietary modification of this format If using X ray film be sure to use the software for your flatoed desktop scanner to convert the image of the film into this format NOTE Appearance of Array Image Description of Control Features All Oligo GEArrays contain a pre biotinylated oligonucleotide with an artificial sequence BAS2C in your gene table and lightly circled in Figure 5 in the lower right hand corner of the membrane if oriented as described above Spots immediately to the left of the corner contain smaller amounts of the sa
14. be changed from 60 C to 37 C and the lid left open while the GEArrays continue to shake on the HybPlate platform Once the thermoshaker temperature reaches 37 C the lid should be closed again iv Shaking speed settings 1 GEArray Express microarrays require small displacement rapid agitation for optimal performance Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 11 e A The GEArray Express Thermoshaker should be set for 1200 rpm for all incubation steps v Securing Multi Chamber HybPlates to the shaker platform 1 2 HybPlates MUST be firmly clamped into the thermoshaker platform to avoid loss of samples and inconsistent processing The clamp screw knob does not need to be completely unscrewed to place or remove HybPlates from the platform It is only necessary to loosen the knob until the clamp band can be rotated 90 and off the covers of the HybPlates c Using the GEArray Multi Chamber HybPlate and seal GE Array Multu Chamber HybPlates are single use hybridization vessels i Positioning of GEArrays within HybPlate 1 2 9 GEArrays should only be handled with smooth flat forceps which should only touch the edges NEVER TOUCH THE PROBE SPOTS A separator paper has been placed between each GEArray and should be discarded when the GEArray is transferred into the HybPlate DO NOT place the plastic li
15. d onto the tray until the GEArrays have been pre moistened with water because static interactions may cause the dry arrays to jump up and cling to the lid Fewer than 8 GEArrays can be hybridized in a GEArray Multi Chamber HybPlate however the HybPlate can only be used for one cycle of hybridization and detection After the detection phase GEArrays can be photographed directly in the HybPlate Therefore when a CCD camera is to be used for the image acquisition phase the position of the GEArrays within the HybPlate should kept as compact as possible in order to provide as many pixels as possible for each GEArray image See Figure 4 for details This consideration in not required for X ray film or scanning as the GEArrays must be removed from the HybPlate for accurate imaging li HybPlate cover and GEArray Multi Chamber Seal 1 The clear plastic cover for the HybPlate provides protection to the GEArrays during processing It must be placed on the HybPlate before securing the HybPlate onto the thermoshaker even when the Multi Chamber Seal is applied The Multi Chamber Seal is a self adhesive foil designed to adhere to the top edges of the HybPlate providing a liquid tight barrier between adjacent hybridization chambers This is necessary to avoid any cross contamination of Target Hybridization Mix Because the adhesive covers only a limited portion of the foil surface the protective paper covering the adhesive should peeled off slowly t
16. eotides are insured to be gene specific using a proprietary computer alogrithm The Oligo GEArrays are the result of combining superior array design with state of the art arraying and detection technologies They are easy to use sensitive economical and accessible for routine use in every research laboratory SuperArray s new GEArray Express System takes our Oligo GEArray to new levels by offering a substantially faster and easier handling method for obtaining GEArray expression analysis data Combined with the TrueLabeling AMP 2 0 cRNA labeling kit and SuperArray s new GEArray Express Thermoshaker and Multi Chamber HybPlate it is now possible to go from total RNA sample to GEArray image within a single day The GEArray Express microarray contains from 96 to 440 oligonucleotide probes representing genes associated with a specific biological pathway The probes are printed on a 2 5 x 3 8 cm nylon membrane with an advanced non contact printing technology in rows of 8 12 or 16 columns A unique bar coded serial number is printed on each membrane for easy tracking and documentation The GEArrays are designed for one time use only Each array membrane is packaged stored and used its own disposable hybridization tube Arrays can be purchased in any quantity allowing for a parallel and comparative study of multiple samples Finally our web based completely integrated image and data analysis software the GEArray Expression Analysis
17. er than 8 arrays every chamber must be filled for uniform cooling Dilute an aliquot of the AP Streptavidin stock 1 8000 in GEAblocking Solution Q 2ul into 16 ml at room temperature This step should be performed just before the second Wash 2 incubation is completed v Make sure the thermoshaker has cooled below 45 C before proceeding with the next step If the thermoshaker has not reached 45 C by this time continue the Wash Solution 2 incubation until the it does vi Pour off the Wash Solution 2 and tap the HybPlate on paper towels b AP Streptavidin incubation and washes i Add 2 ml dilute AP Streptavidin to each GEArray and incubate for 10 shaking at 37 C then pour off as above li Wash with 2 ml room temperature 1X Buffer F shaking at 37 C for 5 and pour off as above iii Repeat the above step 3 more times c Enzyme buffer equilibration and CDP Star incubation i Add 2 ml room temperature Buffer G and incubate for 1 minute If running more than 1 HybPlate AND using a CCD camera system remove the HybPlates to room temperature and hold at this step Proceed with the next step one plate at a time ii Pour off Buffer G as above add 1 0 ml CDP Star and incubate shaking at 37 C for 5 minutes then acquire the GEArray chemiluminescent image using one of the following methods 1 Pour off the CDP Star tap the inverted tray on paper towels and photograph the arrays in the HybPlate without the lid using a high
18. grounds and good dynamic ranges In addition a CCD camera eliminates requirement for direct handling of GEArrays for image acquisition A number of different vendors provide suitable image stations e g Alpha Innotech Syngene Kodak and UVP In the absence of a CCD camera image station certain types of phosphor imagers also detect the chemiluminescent array image e g Typhoon by GE Healthcare Alternatively the array image can also be recorded using X ray film and a flatoed desktop scanner but with a much narrower dynamic range b Exposure Time Overall array image intensity varies from day to day and from person to person making an initial prediction of the exposure time difficult As a general rule the exposure time for capturing the array image when using either a CCD camera or a X ray film should produce the largest possible dynamic range in the individual signals In other words the exposure should obtain the maximal difference between the blank spots with no array signal and the brightest spots with maximal array signal that can be achieved using the medium of your choice For the first time users we recommend starting with an initial exposure of 1 2 min on X ray film or 20 min exposure using a CCD camera system Adjust the exposure time after and based on the initial recording We recommend collecting exposures of the chemiluminescent signals within 15 minutes of the CDP Star treatment to minimize the intensity of the non specific b
19. lso be increased up to 1 12 000 3 Improper incubation time with AP streptavidin The incubation should be no longer than 10 minutes but it may also be reduced 4 Improper washing temperature conditions Be sure to use 2 0 ml Wash solution 2 during the high stringency washing step at 60 C 5 Biotin contamination in containers or buffers Milk contains a high amount of biotin Never use any lab ware that has been used for Western blotting 7 Overexposure Try multiple exposures for various shorter times 8 Too much total RNA or labeled CRNA target Reduce the amount of total RNA or used for CRNA Target Synthesis and Labeling or reduce the amount of labeled CRNA used for hybridization C have performed the pre hybridization of the array but my CRNA synthesis efficiency is poor Can save the membrane until I try the assay again Yes Rinse the GEArray with RNase free water and allow to dry at room temperature Once you define and correct the problem and repeat the RNA isolation and or CRNA synthesis reaction repeat the pre hybridization of the array with fresh GEAprehyb pre warmed to 60 C E Can I strip and re probe the membranes The Oligo GEArray is designed for one time use only Stripping and re probing is not recommended We found that stripping results by individual users varies widely and even under the best conditions 80 percent of the original signal is lost despite the UV crosslinking of the oligonucleotides t
20. me oligonucleotide These spots are detection controls If the detection steps were performed properly these spots should always appear in this pattern All Oligo GEArrays also contain a specific set of housekeeping genes darkly circled in Figure 5 which usually have a relatively high level of basal expression All of these spots can be used as landmarks to insure that the array image has been placed in the correct orientation for data analysis Figure 5 Sample raw images from low density left catalog numbers 001 400 intermediate density middle catalog numbers 401 800 and high density catalog numbers 801 900 Oligo GEArrays are displayed The housekeeping gene landmarks are darkly circled and the biotinylated artificial sequence landmarks in the lower right hand corners are lightly circled These orientations with gene table position number one in the upper left hand corner are optimal for data acquisition and analysis with the GEArray Expression Analysis Suite software Use our specially designed web based and completely integrated GEArray Expression Analysis Suite to complete your data analysis Refer to the software s User Guide for Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 18 Se more information and further instructions The software is a web based application run from a different address than our company s web site and
21. ny s web site and requires a separate log in ID and password obtainable upon ordering a subscription to the software If you haven t already please be sure to place an order for the software with a Customer Service representative Trizol is a registered trademark of Molecular Research Center Inc Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 Bo IV GEArray Express Process Please read through this entire protocol before beginning your experiment RNA samples and cRNA target are very sensitive to RNase digestion therefore wear gloves and maintain an RNase free work area while performing this protocol A RNA Preparation and Quality Control High quality sample RNA is ESSENTIAL for obtaining good microarray results The most important prerequisite for any gene expression analysis experiment is consistent high quality RNA from every experimental sample before starting the labeling process Therefore the sample handling and RNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the RNA or decrease the efficiency of if not block completely the enzyme activities necessary to produce amplified and labeled CRNA of consistent quality for microarray analysis 1 Recommended RNA Preparation Methods High quality total RNA for your microarray experimen
22. o avoid folding the seal back onto itself Additionally because of the shape of the adhesive on the foil care should be taken to accurately align the Multi Chamber Seal over the top edges of the HybPlate with the plastic cover removed before pressing the seal into place To ensure complete contact along all the HybPlate edges and the Seal a soft cover book or catalog should be used to firmly press the Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 12 Se entire Seal against the HybPlate This should be done before placing the plastic cover on the HybPlate speeeseeoeowersov EMM UMM 8 Se ee ees pea Sees ee ees bt bv TEMG eh ES er IMM GN Figure 4 Proper positioning 4 Oligo GEArrays in the GEArray Multi Chamber HybPlate for CCD camera photography of the microarrays Panel A displays one example of compactly positioned GEArrays that will allow optimal photographic framing Panel B shows a configuration that would create a large empty space pixels that are not displaying microarray probe intensities within the image when framed to photograph all the arrays in one image iii Multi Chamber HybPlate buffer removal techniques Two different methods are employed to remove solutions from the hybridization chambers in the HybPlate Aspiration is used when it is important to kee
23. o the nylon membrane Stripping and re probing may be attempted under extreme circumstances or for troubleshooting purposes but the subsequent results will not be publication quality Strip the membranes using a procedure similar to stripping blots used for Northern or Southern hybridization Boil for 5 to 10 minutes in 0 5 SDS cool for 10 minutes and rinse twice with 2X SSC Store the damp membrane at 20 C in its original plastic tube if needed If you have additional questions please check our website www superarray com for a more complete listing of Frequently Asked Questions FAQs or call our technical support representatives at 0800 2466651 D or 49 40 853260 23 27 37 Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 20 Se Express System BIOMOL GmbH Waidmannstr 35 m 22769 Hamburg O biomol info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 449 40 85326022 or 0800 2466652 D Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com
24. obtain enough CRNA to perform the GEArray Express analysis However the RNA may need to be concentrated by ethanol precipitation to at least 11 ng ul before use in the following protocol See the TrueLabeling AMP 2 0 Kit User Manual for more details B TrueLabeling AMP 2 0 CRNA Synthesis and labeling Conversion of experimental RNA to labeled CRNA target see the TrueLabeling AMP 2 0 Kit GA 030 User Manual for this part of the GEArray Express protocol C GEArray Express Hybridization Protocol 1 Handling Considerations a Uniformity of handling conditions from sample preparation to image analysis variable selection are required to produce consistent microarray results Investigators should strive to perform all technical manipulations within this protocol accurately and uniformly b Using the GEArray Express Thermoshaker i The GEArray Express Thermoshaker is required for the hybridization and detection phases of the GEArray Express System ii The GEArray Multi Chamber HybPlate with GEArrays in it should always be kept on the heated platform except when actually being manipulated iii Temperature control settings 1 The thermoshaker should be plugged in and initially set to 60 C 2 The heated lid should be kept closed except during HybPlate manipulation steps and cooling the thermoshaker to 37 C 3 During the temperature transition between the hybridization and detection phases the temperature setting should
25. ocessing or it can be broken down into two major operations CRNA preparation and GEArray hybridization which can be performed on different days at the user s convenience However once the Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 13 Se hybridization process is initiated the entire protocol must be completed on schedule in order to produce consistent microarray data Variations in hybridization or wash times can alter the probe signal pattern affecting the final expression profile analysis li When labeling and hybridizing in one day the only step that may be extended with risk of significantly affecting the GEArray results is the prehybridization step which may be performed for longer than 30 minutes Doing so allows the GEArray wetting and prehybridization steps to be performed during the TrueLabeling AMP 2 0 CRNA synthesis incubation The prehybridization then takes place simultaneously with the CRNA purification and quantification phases allowing the hybridization to start immediately after the cRNA QC assessment 2 Preparation Phase a Wash buffer preparation I Wash Solution 1 2X SSC 1 SDS Mix 10 ml 20X SSC 5 ml 20 SDS and 85 ml ddH2O li Wash Solution 2 0 1X SSC 0 5 SDS Wash Solution 2 is a high stringency wash buffer ts salt concentration is very critical for good GEArray results Prepare it with extra care
26. or Other Biological Samples Refer to existing literature to find isolation protocols for high quality RNA from other biological samples Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 Oo 2 RNA Quality Control For best results from this kit all RNA samples should be suspended in RNase free water not DEPC treated water or RNase free 10 mM Tris buffer pH 8 0 All RNA samples must also meet all of the following criteria before proceeding with this protocol a RNA Concentration and Purity by UV Spectrophotometry Measured in RNase free 10 mM Tris pH 8 0 buffer i Concentration by Azgo should be greater than 11 ng ul total RNA li Azeo Azg0 ratio should be greater than 2 0 iii Azgo A230 ratio should be greater than 1 7 b Ribosomal RNA band integrity Electrophorese a fraction of each RNA sample on a denaturing agarose gel or on an Agilent BioAnalyzer using an RNA 6000 Nano LabChip and verify that there is a sharp distinction at the small side of both the 18S and 28S ribosomal RNA rRNA bands or peaks Any smearing or shoulder to the rRNA bands or peaks indicates that degradation has occurred in the RNA sample MDA231 1 18S 28S 50 o A IN ia x a A 20 25 30 35 40 45 s0 55 5 Figure 3 Good Ribosomal RNA Band Integrity Is Important for Best Results from TrueLabeling AMP 2 0
27. p the solution from contaminating adjacent chambers hybridization or to keep the edges of the HybPlate dry for good sealing to the Multi Chamber Seal wetting and prehybridization Pouring is used for quick easy removal of buffers when cross chamber contamination is not important washing and detection steps 1 Aspiration can be performed using any standard laboratory aspiration system such as a Pasteur pipet attached to a vacuum pump and liquid trap or a water vacuum system or even a serological pipet and Pipet Aid During the aspiration of buffers the tip of the pipet should be placed into one corner of the HybPlate chamber It is important to use low level vacuum to minimize the risk of the tip of the pipet grabbing the microarray membrane which can created artifacts on the final microarray image Avoid touching the aspirator tip to the probe spotted region of the GE Array 2 Pouring off buffers should be done so that the buffer flows slowly out of one of the HybPlate corners and care should be taken to avoid pouring the array off with the buffer After the HybPlate is empty it is then inverted and gently tapped on 2 or 3 paper towels to absorb any residual drops left on the hybridization chamber walls The surface tension of the buffer keeps the GEArray stuck to the bottom of the hybridization chamber d Procedural timing and mid protocol break points i The GEArray Express System is designed to offer either 1 day pr
28. requires a separate log in ID and password obtainable upon ordering a subscription to the software If you haven t already please be sure to place an order for the software with a Customer Service representative V TROUBLESHOOTING GUIDE AND FREQUENTLY ASKED QUESTIONS A WEAK OR NO SIGNALS Low signal intensity is an indication of poor RNA starting material quality RNA quality is crucial to the success of the array Double check the method of RNA isolation used as well as the quality and integrity of your experimental RNA as described If the RNA has been prepared improperly or if there is any evidence of poor RNA quality perform your RNA preparation again before repeating the array analysis Other than RNA sample quality the following factors may also impair your results 1 Low RNA sample concentration If the RNA sample is too dilute it may not perform well in the assay Concentrate your total RNA to at least 11 ng ul or up to 0 5 mg ml using a standard ammonium acetate ethanol precipitation based method 2 Inhibited CRNA Target Synthesis a Total RNA should be dissolved in RNase free dH2O or RNase free 10 mM Tris buffer pH 8 0 Do not use DEPC treated water b If CsCl LiCl guanidinium or organic extractions were used in the process of your total RNA purification trace amounts of these contaminants will inhibit CRNA target synthesis Clean up the RNA with our ArrayGrade Total RNA Isolation Kit SuperArray Catalog GA 013
29. s These dots fade by the end of the procedure when this side of the membrane then appears blank This side of the membrane should always face up inside the multi well hybridization chamber and should face your film or CCD camera during image acquisition Every array also has a unique bar coded serial number to allow tracking of the array during the course of your experiment Before starting your experiment always record each array serial number next to the identity of the sample processed on that array GEArray Express Microarray Printing Format Printed Array Spots and Bar Coded Serial Number appear on the SAME side Printed Array spots ranging from 8 16 gene per row Unique array serial number barcode Blank gt E m Om gt HE n oa Array printing side Reverse side Technical Support 0800 2466651 D 49 40 853260 23 27 37 ts biomol de www SuperArray com GEArray Express User Manual Version 1 0 8 5 2005 Be ll MATERIALS PROVIDED GEArray Express membranes in an ExpressPak box lil ADDITIONAL MATERIALS REQUIRED A Oligo GEArray Trial Kit OR Oligo GEArray Reagent Kit GA 029 or GA 034 Contain enough reagent kits to process up to four or twelve microarrays respectively including the TrueLabeling AMP 2 0 Kit GA 030 the ArrayGrade cRNA Cleanup Kit GA 012 GEAhyb Hybridization Solution H 01 and the Chemiluminescent Detection Kit D 01
30. t must be prepared using one of the following methods each specific for your biological sample Cultured Cells Use our ArrayGrade Total RNA Isolation kit SuperArray Catalog GA 013 Tissue Samples Use a two step protocol First extract RNA from the tissue using the TRIzol protocol Invitrogen Catalog 15596 026 Be sure to use a sufficient amount of TRIZol reagent During homogenization add a volume of reagent at least ten times greater than the tissue volume Then after the ethanol precipitation step further clean up the RNA using our ArrayGrade Total RNA Isolation Kit SuperArray Catalog GA 013 Whole Blood Samples Before RNA preparation red blood cells RBC must be removed from whole blood samples using a density gradient centrifugation medium for example Lymphoprep Greiner Bio One Catalog 1031966 The white blood cell fraction is then used for RNA isolation with our ArrayGrade Total RNA Isolation Kit SuperArray Catalog GA 013 Alternatively the PAXgene Blood RNA Kit Qiagen Catalog 762134 can also be used to prepare total RNA from whole blood samples Total RNA Isolated Using a Phenol Based Method If you have already prepared total RNA from any biological source material using a phenol based method such as TRIzol RNAzol etc you must clean up the RNA with our ArrayGrade Total RNA Isolation Kit SuperArray Catalog GA 013 to insure good target synthesis and good GEArray results F
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