CodeLink Gene Expression System: 16-Assay Bioarray
Contents
1. m e 9 Post hybridization WAS m 9 Detection with streptavidin dye CONJUGATEC cscsccccscscsccccccscsceccccsceccccccecscsccccececececcececeseccecsces 10 and analysis 11 Appendix 1 Stock solutions ees co oda exerc exit 14 Appendix 2 Bacterial mRNA control use interpretation cccccccscscsccccccscsccccccsccccccecscsccccscnceees 16 Appendix 3 Sealing the 16 assay Chamber 17 Process TOW Cl altes vb 18 Ordenpsdnl OFF adtiOltstes dedos eb i mede rA IEEE EPI IR 19 MSS RISO I ULT 20 2 Page CodeLink Expression Bioarray System System description CodeLink Expression Bioarray System comprises a set of bioarray products and tools for gene expression profiling experiments that allows monitoring of the mRNA levels of multiple genes simultaneously The system includes sets of carefully designed and validated bioarrays with integrated hybridization 16 assay chambers that cover a select group of discovery genes for several organisms target preparation and hybridization reagents optimized target preparation and processing proto2. For each reagent required but not provided follow the manufacturer s safety requirements Handling These procedures involve working with RNA therefore exercise great care to avoid RNase contamination All solutions must be RNase free and pipette tips must be aerosol resistant and changed before each step Use commercially prepared nuclease free water rather than water treated with diethylpyrocarbonate DEP C for all nucleic acid steps RNeasy is recommended for total RNA isolation Storage CodeLink iExpress Assay Reagent Kit components should be stored as follows hybridization buffer component A at ambient temperature hybridization buffer component B and 5x fragmentation buffer at 4 The biotin labelled cRNA target is prepared by a linear amplification method The poly A RNA subpopulation within the total RNA population is primed for reverse transcription by a DNA oligonucleotide containing the T7 RNA polymerase promoter 5 to a d T 24 sequence Fig 1 After second strand cDNA synthesis the cDNA serves as the template for an in vitro transcription IVT reaction to produce the target cRNA The IVT is performed in the presence of biotinylated nucleotides to label the target cRNA This method produces approximately 1000 fold to 5000 fold linear amplification of the input poly A RNA A set of bacterial control mRNAs is included in CodeLink iExpress Assay Reagent Kit as controls for the cDNA synthesis and the I
3. high background can be removed by rerinsing Refer to the manufacturer s manual for use and maintenance of the scanner 1 7 14 In the Load and Scan Slide screen the standard TIF file name for the current bioarray is displayed If desired the name may be changed 7 15 Click Browse to select the image path or the location where the image files will be stored If a Security Alert message box is displayed click Yes 7 16 Click Scan Slide The Image tab will display and the instrument will perform the scan 7 17 When complete the view will return to the Report tab 7 18 Click Save Image to save the scanned image at the appropriate file location 7 19 Slide the cover to the left and remove the bioarray 7 20 To scan the next bioarray click New Slide and enter the serial number for the next bioarray The setting information previously entered will be retained 7 21 Analyze the image from each bioarray using the CodeLink Expression Analysis software 13 Page Appendix 1 Stock solutions 1x TNT buffer 0 10 M Tris HCl pH 7 6 0 15 M 0 0596 Tween 20 1 Prepare sufficient volumes of 1x TNT buffer for the entire procedure A single 16 assay bioarray one slide utilizes approximately 16 ml of 1x TNT 2 Rinse a 500 ml reagent container with 100 ml isopropanol Rinse the reagent container twice with 100 ml of deionized water and completely drain the reagent container 3 For 250 ml in a separate container add the f
4. prepare the centrifuge counter balance unit for the bioarray drying step Caution Move into step 6 5 the drying step immediately because slides may dry non uniformly in the air leading to non uniform signal loss Remember to use a centrifuge balance unit 6 Detection with streptavidin dye conjugate 6 1 Remove sealing strip as indicated in step 5 2 Aspirate the 0 75x TNT ensuring that a fluid layer is kept on the bioarray 6 2 Add 250 ul of Cy5 Streptavidin for Microarrays for Microarrays working solution to each assay chamber Place 16 assay Chamber Unit in a dark area at ambient temperature for 30 minutes 6 3 After the 30 minutes incubation aspirate the staining solution Flush each assay chamber three times with 250 ul of 1x TNT Add 250 ul of 1x TNT to each assay chamber and incubate at ambient temperature for 20 minutes 6 4 Aspirate the 1x TNT Add 250 ul of 0 1x SSC 0 0596 Tween 20 solution s Appendix 1 into each chamber immediately invert the 16 assay Chamber Unit s and decant liquid to waste 6 5 Place a paper towel at the bottom of the holder to collect the liquid Quickly invert each Chamber Unit on the tray Place the tray into centrifuge bucket Dry the bioarrays by centrifugation in the Qiagen Sigma 4 15C centrifuge with corresponding bucket rotor 2 x 96 well plate or similar system using following settings speed 2000 rpm 644 x g acceleration 9 deceleration 9 time 3 minutes 10 Page 6 6 Hold t
5. working solution after incubation 7 Wash Flush each assay chamber three times with 250 ul of 1x TNT Add 250 ul of 1x TNT to each assay chamber and incubate at ambient temperature for 20 minutes Aspirate the 1x TNT and add 250 ul of 0 1x SSC 0 05 Tween into each assay chamber Immediately invert the 16 assay chamber unit and decant liquid into waste 8 Dry slides Quickly place the inverted chamber unit s into the slide drying tray and place into the centrifuge rotor bucket with an absorbent paper towel at the bottom Spin dry the Bioarrays Separate the slide from the chamber unit by removing the unit clips with one hand while holding the associated slide with the other hand Store dried bioarrays in the dark 5 9 Bioarray scanning and analysis Scan bioarrays using the 5 um resolution setting and analyze with the CodeLink Expression Analysis software Scanning with GenePix 4000B is described in this booklet For use of CodeLink Bioarrays withother scanners visit our website www appliedmicroarrays com Use only high quality cRNA generated with a CodeLink Expression Assay target preparation protocol for the following steps to achieve optimal results Avoid tip contact with the probe containing area of each bioarray Exercise caution to avoid RNase contamination All solutions must be RNase free pipette tips must be changed before each step Use nuclease free water for all nucleic acid steps Use aerosol res
6. VT reactions These controls are added to the total RNA sample during target preparation Each step of the CodeLink Expression Bioarray processing procedure including target preparation and hybridization can be monitored using these control mRNAs Additionally the bacterial control mRNAs can be used to estimate the sensitivity of RNA detection Hybridization is performed overnight in a temperature controlled shaking incubator Optimized hybridization buffer components are also included in CodeLink Expression Assay Reagent Kit for use at this step Post hybridization processing includes a stringent wash to remove unbound and non specifically hybridized target molecules a staining step with a Cy 5 Streptavidin conjugate and several non stringent washing steps to remove unbound dye conjugate Following a final rinse the bioarrays are dried by centrifugation and scanned Analysis of the bioarrays with the CodeLink Expression Analysis software is described in the Help accessible in the software 4 Page CodeLink Gene Expression System 16 Assay Bioarray Hybridization and Detection Protocol overview Note no overage is included in the amounts described in this Overview 1 Fragment cRNA 16 arrays require 96 ul total volume For each array bring 0 9 ug of cRNA to a final volume of 4 8 ul with water Add 1 2 ul of 5x fragmentation buffer and incubate at 94 9C for 20 minutes Cool 5 minutes 2 Prepare hybridization reaction mixture Br
7. at maximum speed Incubate the hybridization solution at 90 9C for 5 minutes to denature the cRNA 2 4 Cool the tube s on ice for at least 5 and no more than 30 minutes Load all bioarrays within 30 minutes of denaturing the cRNA 8 Page 3 Loading of reaction mixtures into bioarray chambers 3 1 Set the 12 unit Universal shaker tray on a level surface Place the 16 assay Chamber Unit s into the shaker tray with the openings facing up During array chamber loading take care to avoid cross contamination 3 2 Vortex the hybridization reaction mixture for 5 seconds at maximum speed Centrifuge briefly to gather the liquid at the bottom of the tube Place the tube back on ice 3 3 For each bioarray chamber draw the hybridization reaction mixture 60 ul into a pipette tip Place the pipette tip at an angle above the bioarray resting on the edge of the chamber unit Fig 2 3 4 Expel the liquid into the middle of the chamber taking care to avoid forming bubbles or splashing into other array chambers 3 5 After loading seal the Chamber Unit using the sealing strips and sealing tool as described in Appendix 4 4 Hybridization 4 1 Align the 12 unit Universal shaker tray notches with the front and back posts fixed to the shaker incubator platform to place the loaded shaker tray into the shaker incubator The Chamber Unit should be facing up 4 2 Set the shaker speed to 225 rpm and incubate slides for 18 24 hours at 37 9C It is cr
8. cols hybridization and bioarray processing tools bioarray analysis software This user manual provides the protocol for the CodeLink 16 assay bioarray processing including hybridization post hybridization processing and scanning The 16 assay bioarray format consists of 16 independent arrays and hybridization chambers in the space of a standard glass slide 1 Prepare total RNA and bacterial controls 2 First strand cDNA synthesis 42 C 2 h 3 Second strand cDNA synthesis 15 C 2h 4 cDNA purification 5 viro transcription and biotin labelling of CRNA 37 C 14h cRNA purification Fragment cRNA 94 C 20 min 8 Hybridization 37 C 18 24 h g Wash and stain with fluorophore Total mRMA Spikes Fig 1 CodeLink Expression Bioarray signals are produced by the hybridization of biotin labelled complementary RNA cRNA target s to DNA oligonucleotide probes attached to a gel matrix followed by secondary labelling and signal detection 3 Page Safely wamings and precautions Consider all chemicals as potentially hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Wear suitable protective clothing such as laboratory coats safety glasses and gloves Exercise caution to avoid contact with skin or eyes if contact should occur wash immediately with water See Material Safety Data Sheet for specific recommendations
9. dicated by the red line and the positive control probe signal above that line shows that the ability to detect signal above noise is at an estimated sensitivity of one copy per cell Good system performance is indicated if gt 70 of the positive controls probes exhibit signal above their local noise Control Probe Graph Probe 100 e E c D th 5 C E c Ga c qM e a f 4 A Da ATA AS Aa A AR RS en 49 NS ee Poste Con Fig 2 Typical sensitivity plot showing that normalized signal from each bacterial control probe is above noise for the set of control mRNAs spiked at an estimated mass ratio of 1 300 000 16 Appendix 3 Sealing the 16 assay Chamber Unit 1 Place the Sealing Strip over the 16 assay Chamber Unit using the sealing tool Adhesive will attach firmly at first contact Use caution to ensure that the Sealing Strip is in the proper orientation before placing it on the chamber Do not remove a misaligned Sealing Strip Instead place a second strip to cover any open ports 17 Page Appendix 4 Process flow charts 1 Hybridization and Bioarray Processing Fragment cRNA add 5 Fragmentation Buffer 94 C 20 min Prepare Hybridization Mix mix f
10. en buffer is needed for a single slide of 16 arrays 2 In a clean 500 ml container add the following 248 6 ml deionized Water 125 ul Tween 20 1 25 ml 20x SSC buffer Mix well by swirling This solution can be stored up to 2 weeks at ambient temperature 15 Page Appendix 2 Bacterial mRNA control use interpretation Extracting Positive Control Data from the CodeLink Expression Analysis Software On the Expression Analysis menu in CodeLink Expression Analysis v5 0 select Control Probes Report Select and use the Positive Controls with spots of all quality flags and update the graph The graph can be easily copied and pasted for record keeping purposes Interpretation The final mass ratios of the bacterial control mRNAs to the total RNA for the dilutions given are 1 10 000 000 for araB entF fixB gnd hisB and leuB For a typical mammalian cell mRNA comprises 1 596 of the total RNA content If the proportion of mRNA content within total RNA for a particular tissue is unknown then an estimate of the mass ratios of the bacterial control mRNAs to the target mRNA within the total RNA can be made by assuming a midpoint of this range or 396 mRNA content The above total mass ratio then corresponds to an estimated mRNA mass ratio of 1 300 000 for all the bacterial transcripts The normalized signals from these controls can be graphed generating sensitivity plots like that shown in Figure 2 below The noise level for each probe is in
11. ents To mix pipette gently up and down or vortex with moderate speed Do not allow foam to form Place on ice for 5 minutes Repeat the mixing three additional times placing on ice between mixings Protect this solution from light at all times Minimize exposure of Cy5 containing solutions to ambient light 2 Aliquot the Cy5 Streptavidin for Microarrays solution for the number of bioarrays processed in a typical run For example dispense 900 ul aliquots for 3 separate slides 48 arrays 3 This solution can be stored up to six months or until stock bottle expiry date at 20 9C protected from light Once thawed unused portions of aliquots may be used for up to one week if stored protected from light at 4 C Avoid multiple freeze thaw cycles 1 500 Cy5 Streptavidin for Microarrays working solution 1 Prior to dilution centrifuge the thawed Cy5 Streptavidin for Microarrays stock for 1 minutes at 2 8000 x g to remove any precipitates 2 Add 8 8 ul of the Cy5 Streptavidin for Microarrays stock solution to 4 4 ml of filtered TNB buffer for each 16 assay Chamber Unit to be processed Mix gently by inversion Use this working solution within 15 minutes of preparation 0 1x SSC 0 0596 Tween 20 1 liter 20x SSC Ambion 9763 Tween 20 1 Make sure all solutions are molecular biology grade Prepare sufficient volume of 0 1x SSC 0 0596 Tween buffer for the entire rinsing or re rinsing procedure Approximately 4 ml of 0 1X SSC 0 0596 Twe
12. er power 10096 pixel size 5um focus position O um 7 10 In the Report tab open the scanning script by clicking Scan CodeLink Slide 7 11 Enter the bioarray serial number and click Next The Experiment and Scan Information interface is displayed 11 Page 7 12 in the project name experiment name and sample name The username is automatically captured When opening this interface for the first time a message box may ask whether to allow an ActiveX interaction to proceed Click Yes 7 13 Select a setting gps file The standard setting file is CodeLinkExpr gps If a settings file was previously selected the name and path are displayed under Current Settings File To select a new file click Browse under Select New Settings File The values for user name and settings file that were entered for a previous bioarray are retained but may be changed 12 Page Ensure that the slide does not move laterally over the clear gasket this will damage the array surface Keep the bioarrays covered until they are scanned to protect them from dust and light and handle them only with gloves For best results scan bioarrays within two days of staining If bioarrays will be retained for future scanning they should be stored in a dry box protected from light If regions of high background on the bioarray are noted upon scanning the bioarrays may be rerinsed by repeating steps 6 4 and 6 5 However not all incidents of
13. he Chamber Unit with chamber side away from you Remove the unit clips with one hand while holding the slide with the other Peel the slide away from the Chamber and place the dry bioarrays into a light protected slide box until they are scanned Bioarrays should be scanned within two days of assay completion 6 7 Wash the slide drying tray with Alconox soap Rinse thoroughly with deionized water to remove residual soap then air dry 7 Bioarray scanning and analysis For scanning with a GenePix Array Scanner use the following steps For information on use of alternative scanners with CodeLink bioarrays visit our website www appliedmicroarrays com 7 1 Turn the scanner on 15 minutes prior to use 7 2 Slide the cover to the left to expose the slide holder 7 3 Lift the latch of the slide holder and lift the upper clip 7 4 While wearing gloves load the bioarray into the tray with the slide number label side down and closest to the front of the scanner 7 5 Pull the clip on the left of the slide out and let the bioarray fall into place Release the clip to put pressure against the bioarray 7 6 Handle the bioarray by the edges and move the bioarray toward you 7 7 Lower upper clip and press down on the latch until it clicks 7 8 Slide the cover to close the slide holder 7 9 Open the GenePix software and select the following settings setting file name Expression 635 16UP 5um gps wavelength 635 nm PMT voltage 600 V las
14. ing 6 ul of the fragmented cRNA solution 18 ul of hybridization buffer component A and 30 ul of hybridization buffer component B to a final volume of 60 ul with water Incubate at 90 9C for 5 minutes and immediately chill on ice for 5 minutes then proceed to loading 3 Loading reaction mixtures into array chambers Slowly pipet the 60 ul hybridization reaction mixture into the middle of each array chamber taking care against splash contamination Seal the entire 16 assay unit with a sealing strip 4 Hybridization Set the shaker speed to 225 rpm and incubate slides for 18 24 hours at 37 9C maintaining a consistent hybridization time for comparative experiments 5 Post hybridization wash Carefully remove the 16 assay Chamber sealing strip Flush each assay chamber three times with 250 ul of 0 75x TNT Add 250 ul of 0 75x TNT to each assay chamber and seal entire 16 assay Chamber unit with a sealing strip Transfer the 16 assay unit to a 46 C oven and incubate for exactly 1 hour Do not exceed 1 hour incubation 6 Detection with streptavidin dye conjugate Remove the 16 assay Chamber unit from the oven and carefully remove the sealing strip Aspirate the 0 75x TNT keeping a small volume of fluid on the slide surface at all times Add 250 ul of Cy5 Streptavidin for Microarrays working solution to each assay chamber Place 16 assay Chamber unit in a dark area at ambient temperature for 30 minutes Aspirate the Cy5 Streptavidin for Microarrays
15. instructions M APPLIED MICROARRAYS CodeLink Gene Expression System 16 Assay Bioarray Hybridization and Detection Warning For research use only Not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals 1 Page Page finder SvsteltixdescripEbION 3 Safety Warnings and v suse vnd deiude cu NV A vo oue ona USOS USUS 4 Storage and Nandline conditiolSassses ede dS uae Cxue nh Urea uua a 4 CodeLink Gene Expression System 16 Assay Bioarray Hybridization and Detection BIOLOCOLDVBEVIPM a ve OPERE VUE 5 Products used TOF DroCcessiB Aue ede aa a 7 Other materials required for processing vix bre Seo E EI 7 Protocol tor hybridization and detecto Yin oS 8 Fragmentation OL CRNA assets sy d vs 8 Preparation of hybridization reaction mixtures sccsccccsceccccsccecsccccsccccsceccscescececcscececcsceccsceccscess 8 Loading of reaction mixtures into bioarray chambers eese eee eee ee eee nennen nennen 9
16. istant tips for all pipetting steps To avoid fluorophore photobleaching prepare the Cy5 labelled Streptavidin conjugate in opaque or dark tubes In addition minimize bioarray exposure to ambient light particularly after completing step 6 2 Ensure that the ambient temperature in the laboratory 5 23 9C 2 9C Minimize exposure to particulate matter Clean all work areas prior to hybridization and analysis 6 Page The list of reagents and equipment is recommended for best performance Contact Applied Microarrays Inc technical support for information on use of alternatives Storage Keep 5x fragmentation buffer at 4 C until use Briefly vortex and spin down prior to use to ensure solution homogeneity Store hybridization buffer component at room temperature Since a precipitate may form upon storage briefly vortex and spin down prior to use to ensure solution homogeneity If necessary warm at 37 to redissolve Store hybridization buffer component B at 4 C until use For steps 1 2 use of a 10 volume mixture overage is recommended but not required cRNA should appear on the gel as a smear If there is no smear or if the smear is predominantly below 500 nucleotides do not continue with the sample 7 Page Products used for processing CodeLink iExpress Assay Reagent Kit Manual Prep 24 reactions 67601000 5x fragmentation buffer hybridization buffer component A hybridization buffe
17. itical that arrays used in any form of comparison are hybridized for the same amount of time within the given range 5 Post hybridization wash 5 1 Remove the Universal shaker tray from the shaker incubator and place on a level surface at ambient temperature Process only 4 Chamber Units of 16 assay slides 64 arrays at a time per person Leave the trays in the shaker until ready to process 5 2 While holding the outside edges of the Chamber Unit firmly remove the 16 assay Chamber sealing strip by lifting the tab and slowly pulling it back at a 60 angle 5 3 Use an 8 channel electronic pipettor set on a medium flow rate and flush each assay chamber three times with 250 ul of 0 75x TNT Add 250 ul of 0 75x TNT to each assay chamber and seal the entire 16 assay Chamber Unit Transfer the unit to a 46 9C oven and incubate for exactly 1 hour longer incubation time may significantly reduce signal intensities 9 Page Filter all TNT buffer solutions Appendix 1 through 0 2 um filter prior to use Prepare sufficient volumes of 1x and 0 75x TNT buffers Appendix 1 for the entire procedure Avoid contact with the probe containing surface of the microarray throughout the post hybridization steps Do notallowthe hioarrays to dry during any of the processing steps Clean the shaker incubator platform and the shaker tray with deionized water after each hybridization run If processing an odd number of Chamber Units during secondary labelling
18. mentation of cRNA 1 1 For each bioarray bring 1 ug of cRNA from step 7 6 of CodeLink target preparation protocol to a volume of 5 3 ul with nuclease free water in a thin walled microcentrifuge tube 1 2 Add 1 3 ul of 5x fragmentation buffer for a total volume of 6 6 ul Place tube in a thermal cycler and heat for 20 minutes at 94 9C using the heated lid feature 1 3 Cool to 9C in the thermal cycler for at least 5 minutes Note Volumes described here include a 10 volume overage Do not exceed 100 ul per tube during fragmentation 2 Preparation of hybridization reaction mixtures 2 1 Set the temperature of the shaker incubator to 37 9C for hybridization Assemble bioarray tray posts from the CodeLink Shaker Kit onto the incubator platform 2 2 Transfer the cRNA sample from thin walled tube to a 1 5 ml microcentrifuge tube For each single array within the 16 assay bioarray slide to be processed prepare 66 ul of hybridization solution by transferring the fragmented cRNA sample into the mixture in the 1 5 ml microcentrifuge tube described below 19 8 ul hybridization buffer component A 33 ul hybridization buffer component B 6 6 ul nuclease free water 6 6 ul fragmented cRNA from Step 1 66 ul total volume If using multiple Chamber Units do not prepare more than 1 ml of hybridization reaction mixture in a single micro centrifuge tube Larger volumes will not allow good heat transfer 2 3 Vortex the solution for 5 seconds
19. ollowing 25ml 1M Tris HCl pH 7 6 7 5 ml 5M 125 ul Tween 20 217 4 ml deionized water 4 Mix well by swirling Filter TNT through a 0 2 um filter and transfer to the isopropanol rinsed reagent container This solution can be stored for up to four weeks at ambient temperature 0 75x TNT buffer 1 Prepare a sufficient volume of 0 75x TNT buffer for the entire procedure Approximately 16 ml of 0 75x TNT is needed per slide 2 Add 25 ml of deionized water to 75 ml of 1x TNT buffer per 100 ml of buffer required TNB Buffer 0 6 liters 0 1 M Tris HCl pH 7 6 0 15 M 0 596 NEN blocking reagent PerkinElmer FP1020 1 Toaclean 2 liter Erlenmeyer flask with a stir bar add the following 522 ml nuclease free water 60 ml 1M Tris HCl pH 7 6 18 5 2 stir plate heated to 60 slowly add 3 g of NEN blocking reagent in 0 5 g increments until all 3 g of blocking reagent are dissolved 3 Turn off heat and continue mixing for 30 minutes While the solution is still warm filter through a 0 88 um filter 4 Aliquot the buffer into 50 ul tubes and store at 20 TNB can be stored for 12 weeks at 20 2C 5 For use thaw TNB overnight at 4 9C Thawed aliquots can be stored at 4 9C up to one week Cy5 Streptavidin for Microarrays stock solution 14 Page 1 Add 1 ml of nuclease free water to the 1 mg bottle of lyophilized Cy5 Streptavidin for Microarrays This reagent already contains buffer compon
20. r component B Cy5 Streptavidin for Microarrays 28900224 staining solution is a 1 500 dilution in TNB see Appendix 1 CodeLink Expression Analysis Software v5 0 310035 CodeLink Universal Shaker Kit 310031 CodeLink Slide 16 Assay Accessory Kit 310032 Other materials required for processing All reagents used in protocol should be molecular biology grade or higher e 0 596 NEN blocking reagent PerkinElmer FP1020 e nuclease free water 1M Tris HCl pH 7 6 e 5 M Tween 20 e20x 55 Ambion 9763 sopropanol to wash reagent container for TNT preparation e GenePix 4000B Array Scanner Axon Instruments and computer configured for GenePix 4000B Array Scanner or other CodeLink validated scanner see www appliedmicroarrays com for CodeLink validated scanner application notes e centrifuge 4 15C Qiagen Corp 81010 or similar with appropriate centrifuge plate rotor 2 x 96 Qiagen Corp 81031 For alternative centrifuges contact Applied Microarrays Inc technical support or visit our Web site pipette tips sterile RNase free and aerosol resistant e 1 5 ml microcentrifuge tubes sterile and RNase free e 0 2 ul thin walled microcentrifuge tubes e pipettors e 8 channel electronic pipettor e microcentrifuge e thermal cycler e 46 9C air incubator e powder free gloves e 50 1200 ul pipet tips e sterile solution basin e water bath 90 9C Protocol for hybridization and detection 1 Frag
21. ragmented cRNA Buffer and Hyb Buffer B Load Slides Carefully pipet ul Mix into each well seal chamber 225 rpm shaker incubator 37 C 18 h Do not allow arrays to dry out during tha following steps Stringent Wash Remove sealing strip 3 flushes with 250 ul 0 75 using 8 channel pipette add 250 0 75 THT in each chamber and seal 46 C 1 h Secondary Labelling Remove sealing strip 1 Flush with 250 nl SA Cy S Staining Solution 220 ul A G Staining Solution ambient temperature 30 min in dark area Wash 3 flushes with 250 ul 1 TNT using 8 channel pipette add 250 1 TNT in each array chamber ambient temperature 20 min in dark area Final Rinse Place up to 3 slides in the Plate Holder remove 1 with 250 ul 0 1 550 0 05 Tween Move immediately to next step Spin Dry Decant fluid from arrays and invert plate holder into centrifuge bucket centrifuge at rcf 3 min remove chamber after spin dry and place slide in a light tight box until scanning Scan Remove slide from Chamber Unit and ecan slide 18 Page References 1 GenePix 4000 User s Guide Axon Instruments Inc 2500 136 Rev E 2001 Applied Microarrays Inc 7700 S River Parkway Tempe AZ 85284 USA Email sales appliedmicroarrays com Phone 480 775 6320 FAX 800 927 9315 www appliedmicroarrays com 19 Page APPLIED MICROARRAYS
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