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One-Color Microarray-Based Prokaryote Analysis

1. way DU TEL TET r 4 H PH LE T 1 Front side Front side ar code left bar code right landscape a landscape THN FUIUL E UL V Agilent Front side bar code down portrait Figure 10 Microarray slide orientation One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Reference 4 Array Sample tracking microarray slides Use the form below to make notes to track your samples on microarray slides Position the gasket slide in the SureHyb chamber base with the label to the left Load the samples top row left to right then lower row left to right The array suffix assignments from Feature Extraction will be in the order shown Arrays Array 1_1 Array 1_2 Array 1_3 Array 1_4 B A R C 0 D E Array 2_1 Array 2_2 Array 2_3 Array 2_4 Barcode Number Figure 11 8 pack microarray slide One Color Microarray Based Prokaryote Analysis FairPlay III Labeling Protocol 71 www agilent com In This Book This guide contains information to run the One Color Microarray Based Prokaryote Analysis protocol Agilent Technologies Inc 2009 2012 2015 Version 2 0 August 2015 G4813 90000 Revision B0 RES Agilent Technologies
2. 10x Gene Expression Blocking Agent 25x Fragmentation Buffer 2x Hi RPM Hybridization Buffer Table 22 Gene Expression Wash Buffer Kit Content Gene Expression Wash Buffer 1 Gene Expression Wash Buffer 2 Triton X 102 10 Table 23 RNeasy Mini Kit Content RNeasy Mini Spin Column pink Collection Tube 1 5 ml Collection Tube 2 ml Buffer RLT Buffer RW1 Buffer RPE RNase Free Water One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 65 4 Reference Supplemental User Guides First time users of the Agilent oligo microarray system please refer to the following user manuals for detailed descriptions and operation recommendations for each of the hardware and software components used in the one color platform workflow The user guides can be downloaded from the Agilent web site at www agilent com chem dnamanuals protocols Agilent Microarray Hybridization Chamber User Guide Hybridization Oven User Manual Microarray Scanner System User Guide G4900DA SureScan Microarray Scanner User Guide Feature Extraction Software Quick Start Guide Feature Extraction Software User Guide Feature Extraction Software Reference Guide 66 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Reference 4 Microarray Handling Tips Each microarray is printed on the side of the glass slide containing the Agilent labeled barcode This si
3. FE Version Tuesday December 01 2009 US90403632 251485049992 S01 14 38 11 GE1_107_Sep09 Read Only nilaguha 10 7 1 1 Sample red green Non Uniform Population pot Finding of the Four Corners of the Arra tex 4A Grid Normal Local Feature Background Green 12 148 Spatial Distribution of All Outliers on the Array Figure 7 532 rows x 85 columns YR nor memes e 1 Tora e E RA d toe Lats o we FeatureNonUnif Green 12 0 03 GeneNonUnif Green 12 0 029 96 BG NonUniform BG Population Green FeaturePopulation e Green Feature NonUniform Green Grid 0 BG Method Background Detrend Multiplicative Detrend Additive Error Saturation Value Net Signal Statistics Agilent SpikeIns Saturated Features 99 of Sig Distrib 50 of Sig Distrib 1 of Sig Distrib Non Control probes Saturated Features 99 of Sig Distrib 50 of Sig Distrib 1 of Sig Distrib Histogram of Signals Plot Page of 3 14850 D F 20090416 No Background On FeatNCRange LoPass True 2 Green 778999 g 0 172449 344 18 Number of Points il 1 0 1 2 3 Log of BG SubSignal Features NonCtrl with BGSubSignal lt 0 ns 4 5 Histogram of Signals 3619 Green Example of the first page of a QC Report generated by Feature Extraction Software One Color Microarray B
4. 68 Array Sample tracking microarray slides 71 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol One Color Microarray Based Prokaryote Analysis Protocol 1 Before You Begin Procedural Notes 8 Safety Notes 9 Agilent Oligo Microarrays 10 Required Equipment 11 Required Reagents 12 Optional Equipment Reagents 13 Required Hardware and Software 13 Optional Software 13 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee microarray performance and does not provide technical support to those who use non Agilent protocols in processing Agilent microarrays E Agilent Technologies d 1 Before You Begin Procedural Notes Determine the integrity of the input RNA for labeling and hybridization prior to use to increase the likelihood of a successful experiment To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips Maintain a clean work area When preparing frozen reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid 3 Store on ice or in a cold block until us
5. Catalytic ozone decomposition filtering system inside the scanner In addition to the ozone barriers the Agilent Stabilization and Drying Solution which is an organic solvent based wash can reduce background variability produced by wash artifacts The use of the Agilent Stabilization and Drying Solution is described in this section For more information visit www agilent com chem dnatechnicalnotes to download the technical note on Improving Microarray Results by Preventing Ozone Mediated Fluorescent Signal Degradation p n 5989 0875EN One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Supplemental Procedures 3 Step 1 Prepare the Stabilization and Drying Solution The Agilent Stabilization and Drying Solution contains an ozone scavenging compound dissolved in acetonitrile The compound in solution is present in saturating amounts and may precipitate from the solution under normal storage conditions If the solution shows visible precipitation warming of the solution will be necessary to redissolve the compound Washing slides using Stabilization and Drying Solution showing visible precipitation will have a profound adverse effect on microarray performance WARNING The Agilent Stabilization and Drying Solution is a flammable liquid Warming the solution will increase the generation of ignitable vapors Do not use an open flame or a microwave Do not increase temperature rapidly Warm and mix the ma
6. For each hybridization do not exceed 20 uL for the total volume of labeled samples see Hybridization on page 29 To reduce the volume continue to spin the sample in a centrifuge and use a concentrator such as a SpeedVac to concentrate the sample One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 6 Quantify the cDNA Quantify the cDNA using NanoDrop ND 1000 UV VIS Spectrophotometer version 3 2 1 CAUTION 1 2 3 Start the NanoDrop software Click the Microarray Measurement tab Before initializing the instrument as requested by the software clean the sample loading area with nuclease free water 4 Load 1 0 to 2 0 uL of nuclease free water to initialize Then click OK 5 Once the instrument has initialized select ssDNA 33 as the Sample type use the drop down menu Make sure the Recording button is selected If not click Recording so that the readings can be recorded saved and printed Failure to engage recording causes measurements to be overwritten with no possibility of retrieval Transfer 1 0 to 2 0 uL of 10 mM Tris base pH 8 5 with a pipette to the instrument sample loading area Click Blank Clean the sample loading area with a laboratory wipe Transfer 1 0 to 2 0 uL of the sample onto the instrument sample loading area Type the sample name in the space provided Click Measure Be sure to clean the sample loading area between measurements a
7. d Add 750 uL of 75 ethanol to the microspin cup Snap the cap of the 2 mL receptacle tube onto the top of the microspin cup Spin the 2 mL receptacle tube in a microcentrifuge at 11 000 to 13 000 rpm for 30 seconds Open the cap of the Z mL receptacle tube remove and retain the microspin cup and discard the wash buffer Repeat entire step 14 Place the microspin cup back in the 2 mL receptacle tube and snap the cap of the 2 mL receptacle tube onto the microspin cup 24 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 15 Spin the 2 mL receptacle tube in a microcentrifuge at 11 000 to 13 000 rpm for 30 seconds When the tubes are removed from the centrifuge make sure that all of the wash buffer is removed from the microspin cup 16 Transfer the microspin cup to a fresh nuclease free 1 5 mL microfuge tube and discard the 2 mL receptacle tube 17 Incubate and spin with 10 mM Tris base a Add 50 uL of 10 mM Tris base pH 8 5 directly above but not touching the fiber matrix at the bottom of the microspin cup b Incubate the tube at room temperature for 5 minutes Maximum recovery of the labeled cDNA from the microspin cup depends on the pH ionic strength and volume of the elution buffer added to the microspin cup the placement of the elution buffer into the microspin cup and the incubation time Maximum recovery is obtained when the elution buffer is lt 10 mM in concentrati
8. 57 3 Supplemental Procedures Step 2 Wash with Stabilization and Drying Solution Use fresh Gene Expression Wash Buffer for each wash group up to eight slides The acetonitrile and Stabilization and Drying Solution can be reused for washing of up to three groups of slides for a total of 24 slides The Stabilization and Drying Solution must be set up in a fume hood Wash 1 and Wash 2 set up areas should be put close to or preferably in the same fume hood Use gloves and eye face protection in every step of the warming procedures 58 Table 18 lists the wash conditions for the wash procedure with Stabilization and Drying Solution Table 18 Wash conditions Dish Wash Buffer Temperature Time Disassembly 1 Gene Expression Wash Buffer 1 Room temperature 1st wash 2 Gene Expression Wash Buffer 1 Room temperature 1 minute 2nd wash 3 Gene Expression Wash Buffer 2 Elevated 1 minute temperature Acetonitrile Wash 4 acetonitrile Room temperature 10 seconds 3rd wash 5 Stabilization and Drying Solution Room temperature 30 seconds The elevated temperature of the second wash step is usually around 31 C due to cooling by the room temperature dish and the rack of arrays 1 Completely fill slide staining dish 1 with Gene Expression Wash Buffer 1 at room temperature 2 Puta slide rack into slide staining dish 2 Add a magnetic stir bar Fill slide staining dish 2 with enough Gene Expression Wash Buffer 1 at room temperature to co
9. Chamber Kit G2534A and can also be downloaded from the Agilent web site at www genomics agilent com Search for G2534A Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base Make sure that the gasket slide is flush with the chamber base and is not ajar Do not let the pipette tip or the hybridization solution touch the gasket walls Allowing liquid to touch the gasket wall greatly increases the likelihood of gasket leakage When you lower the microarray slide on top of the SureHyb gasket slide make sure that the two slides are parallel at all times 2 Slowly dispense 40 uL of hybridization sample onto the gasket well in a drag and dispense manner Position the slides so that the barcode label is to your left Load the samples left to right For 8 pack slides start with the first row The output files will come out in that same order Refer to Array Sample tracking microarray slides on page 71 for guidelines on tracking sample position for multipack slide formats Avoid the introduction of air bubbles to the gasket wells Air bubbles can affect the final sample volume and can cause leakage from the gasket well 3 If any wells are unused a Make a 1x solution of the 2x Hi RPM Hybridization Buffer b Addthe volume of 1x Hybridization Buffer equal to the sample volume to each unused well Make sure all wells contain sample
10. One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 29 2 Procedures Step 2 Prepare hybridization samples 1 For each microarray add each of the components as indicated in Table 11 to a 1 5 mL nuclease free microfuge tube 2 Mix well but gently on a vortex mixer Table 11 Blocking Mix Components Volume Mass cyanine 3 labeled cDNA 600 ng 10x Gene Expression Blocking Agent 5 uL Nuclease free water bring volume to 25 uL Total Volume 25 uL 3 Add 2x Hi RPM Hybridization Buffer See Table 12 Table 12 Hybridization mix Components Volumes per hybridization cDNA from Blocking Mix 25 uL 2x Hi RPM Hybridization Buffer 25 uL 4 Mix well by careful pipetting part way up and down Do not introduce bubbles to the mix The surfactant in the 2x Hi RPM Hybridization Buffer easily forms bubbles Do not mix on a vortex mixer mixing on a vortex mixer introduces bubbles 5 Spin briefly on a microfuge Use immediately Do not store Refer to Microarray Handling Tips on page 67 for information on how to safely handle microarrays 30 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 3 Prepare the hybridization assembly CAUTION Refer to the Agilent Microarray Hybridization Chamber User Guide for more details to load slides and to assemble and disassemble the chambers This user guide is included with the Agilent Microarray Hybridization
11. enzymes are homogenous Immediate after use return components to 20 C Table 10 cDNA Master Mix Component Volume per reaction Volume per 8 reactions includes excess 10x AffinityScript Reaction Buffer clear 2 uL 20 uL cap 20x dNTP Mix with amino allyl dUTP 1 uL 10 uL green cap 0 1 M DTT green cap 1 5 uL 15 uL RNase Block purple cap 0 5 uL 5 uL 7 Add 5 uL of cDNA Master Mix to each sample tube Draw the solution into a pipette and release to mix the solution One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 19 2 20 Procedures 8 Spin the AffinityScript HC Reverse Transcriptase purple cap right before use 9 Add 3 uL of AffinityScript HC Reverse Transcriptase purple cap and incubate at 42 C for 60 minutes 10 Add 10 uL of 1 M NaOH and incubate at 70 C for 10 minutes to hydrolyze RNA 11 Let the sample cool to room temperature slowly Do not cool on ice 12 Spin tube briefly to collect contents 13 Add 10 uL of 1 M HCl to neutralize the solution One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 3 Purify cDNA The cDNA must be purified to remove unincorporated nucleotides buffer components and hydrolyzed RNA Incomplete removal of the Tris and EtOH will result in lower amino allyl dye coupling efficiency Care must be taken to ensure that the pellet is completely dry at the end of step 6 indicating complete
12. or 1x Hybridization Buffer Empty wells can cause failure in hybridization Grip the slide on either end and slowly put the slide active side down parallel to the SureHyb gasket slide so that the Agilent labeled barcode is One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 31 2 Procedures facing down and the numeric barcode is facing up Make sure that the sandwich pair is properly aligned CAUTION Do not drop the array slide onto the gasket Doing so increases the chances of samples mixing between gasket wells 5 Put the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces 6 Firmly hand tighten the clamp onto the chamber 7 Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles If necessary tap the assembly on a hard surface to move stationary bubbles 32 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 4 Hybridize 1 Load each assembled chamber into the oven rotator rack Start from the center of the rack position 3 or 4 when counting from the left Set your hybridization rotator to rotate at 10 rpm when using 2x Hi RPM Hybridization Buffer 2 Hybridize at 65 C for 17 hours CAUTION If you are not loading all the available positions on the hybridization rotator rack be sure to balance the loaded hybridization chambers on the rack so that
13. with the slide rack and stir bar to a magnetic stir plate 3 Fill the staining dish with 100 acetonitrile or isopropyl alcohol Turn on the magnetic stir plate and adjust the speed to a setting of 4 medium speed 5 Wash for 5 minutes Discard the solvent as is appropriate for your site One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 35 2 Procedures CAUTION 7 Repeat step 1 through step 6 8 Air dry the staining dish in the vented fume hood 9 Proceed to Milli Q water wash Milli Q water wash Wash all dishes racks and stir bars with Milli Q water 1 Run copious amounts of Milli Q water through the staining dish 2 Empty out the water collected in the dish 3 Repeat step 1 and step 2 at least 5 times as it is necessary to remove any traces of contaminating material 4 Discard the Milli Q water Some detergents may leave fluorescent residue on the dishes Do not use any detergent in the washing of the staining dishes If detergent is used all traces must be removed by copiously rinsing with Milli Q water 36 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 4 Wash the microarray slides The microarray wash procedure for the Agilent one color platform must be done in environments where ozone levels are 50 ppb or less For Scanner C and Scanner B if ozone levels exceed 50 ppb in your laboratory use the Agilent Ozone Bar
14. 00DA SureScan Microarray Scanner System User Guide Figure 3 Slide in slide holder for SureScan microarray scanner For Agilent Scanner B or C only In environments in which the ozone level exceeds 50 ppb immediately put the slides with active microarray surface with Agilent labeled barcode facing up in a slide holder Make sure that the slide is not caught up on any corner Put an ozone barrier slide cover on top of the array as shown in Figure 4 Refer to the Agilent Ozone Barrier Slide Cover User Guide p n G2505 90550 included with the slide cover for more information As an alternative use the Stabilization and Drying Solution See Preventing Ozone Related Problems on page 56 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 39 2 Procedures Step 4 Wash the microarray slides Figure 4 Inserting the ozone barrier slide cover shown for Scanner B and Scanner C In environments in which the ozone level is below 50 ppb put the slides with Agilent barcode facing up in a slide holder 15 Scan slides immediately to minimize the impact of environmental oxidants on signal intensities If necessary store slides in orange slide boxes in a nitrogen purge box in the dark 40 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Scanning and Feature Extraction Step 1 Scan the slides Agilent provides support for Agilent microarrays scanne
15. 15 Sample Preparation 18 Step 1 Dilute Spike in Solution 18 Step 2 Synthesize cDNA 19 Step 3 Purify cDNA 21 Step 4 Label the modified cDNA 22 Step 5 Purify the dye coupled cDNA 23 Step 6 Quantify the cDNA 27 Hybridization 29 Step 1 Prepare the 10x Blocking Agent 29 Step 2 Prepare hybridization samples 30 Step 3 Prepare the hybridization assembly 31 Step 4 Hybridize 33 Microarray Wash 34 Step 1 Add Triton X 102 to Gene Expression wash buffers 34 Step 2 Prewarm Gene Expression Wash Buffer 2 35 Step 3 Prepare the equipment 35 Step 4 Wash the microarray slides 37 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Contents Scanning and Feature Extraction 41 Step 1 Scan the slides 41 Step 2 Extract data using Agilent Feature Extraction Software 44 Supplemental Procedures 51 Quality Assessment of Template RNA 52 Step 1 Prepare for quality assessment 53 Step 2 Assess the quality using the Agilent 2100 Bioanalyzer 54 Preventing Ozone Related Problems 56 Step 1 Prepare the Stabilization and Drying Solution 57 Step 2 Wash with Stabilization and Drying Solution 58 Normalizing Agilent One Color Microarray Data 61 To do downstream analysis of Agilent microarray data 61 To use Feature Extraction 62 4 Reference 63 Kit Contents 64 Supplemental User Guides 66 Microarray Handling Tips 67 General Microarray Layout and Orientation
16. 7 2 38 Procedures 5 Prepare the hybridization chamber disassembly a Put the hybridization chamber assembly on a flat surface and loosen the thumbscrew turning counterclockwise b Slide off the clamp assembly and remove the chamber cover c With gloved fingers remove the array gasket sandwich from the chamber base by grabbing the slides from their ends Keep the microarray slide numeric barcode facing up as you quickly transfer the sandwich to slide staining dish 1 d Without letting go of the slides submerge the array gasket sandwich into slide staining dish 1 containing Gene Expression Wash Buffer 1 6 With the sandwich completely submerged in Gene Expression Wash Buffer 1 pry the sandwich open from the barcode end only a Slip one of the blunt ends of the forceps between the slides b Gently turn the forceps upwards or downwards to separate the slides c Let the gasket slide drop to the bottom of the staining dish d Grasp the top corner of the microarray slide remove the slide and then put it into the slide rack in the slide staining dish 2 that contains Gene Expression Wash Buffer 1 at room temperature Transfer the slide quickly so avoid premature drying of the slides Touch only the barcode portion of the microarray slide or its edges 7 Repeat step 4 through step 6 for up to seven additional slides in the group For uniform washing do up to a maximum of eight disassembly procedures yielding eight microarra
17. Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 23 2 Procedures 7 Use the pipette to transfer the mixture to the microspin cup that is seated in the 2 mL receptacle tube Do not touch the pipette to the matrix in the microspin cup as you transfer the mixture 8 Snap the cap of the 2 mL receptacle tube onto the top of the microspin cup To ensure proper sample flow use the 2 mL receptacle tube that is provided with the microspin cup Do not substitute another tube 9 Spin the 2 mL receptacle tube in a microcentrifuge at 11 000 to 13 000 rpm for 30 seconds The labeled cDNA is retained in the fiber matrix of the microspin cup 10 Open the cap of the 2 mL receptacle tube remove and retain the microspin cup and discard the DNA binding solution that contains the uncoupled dye 11 Combine 100 uL of the DNA binding solution and 100 uL of 80 Sulfolane mixture Mix well on a vortex mixer Make sure that the two solutions are well mixed before use 12 Wash with DNA binding solution and 80 Sulfolane mixture a Add the 200 uL of DNA binding solution and Sulfolane mixture to the microspin cup Snap the cap of the 2 mL receptacle tube onto the top of the microspin cup Spin the 2 mL receptacle tube in a microcentrifuge at maximum speed for 30 seconds Open the cap of the Z mL receptacle tube remove and retain the microspin cup and discard the DNA binding sulfolane mixture 13 Wash with 75 6 ethanol
18. One Color Microarray Based P karyote Analysis Protocol For use with Agilent Gene Expression oligo microarrays Version 2 0 August 2015 Before you begin view hands on videos of SurePrint procedures at Microarrays manufactured with Agilent SurePrint http www agilent com genomics protocolvideos Technology For Research Use Only Not for use in diagnostic procedures E Agilent Technologies Notices Agilent Technologies Inc 2009 2012 2015 No part of this manual may be reproduced in any form or by any means including electronic storage and retrieval or transla tion into a foreign language without prior agreement and written consent from Agi lent Technologies Inc as governed by United States and international copyright laws Manual Part Number 64813 90000 Edition Version 2 0 August 2015 Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgements Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated Technical Support Technical product support may be obtained by contacting your local Agilent Support Services representative Agilent s world wide sales and support center telephone numbers can be obtained at the following web site www agilent com chem contactus or send an e mail to genomics Jagilent com Warranty The material contained in this docu ment is provided as is and is sub ject to being changed w
19. ased Prokaryote Analysis FairPlay Ill Labeling Protocol 49 50 This page intentionally left blank One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol One Color Microarray Based Prokaryote Analysis Protocol e ee 3 amp Supplemental Procedures Quality Assessment of Template RNA 52 Preventing Ozone Related Problems 56 Normalizing Agilent One Color Microarray Data 61 The procedures in this chapter are supplemental to the main protocol E Agilent Technologies 51 3 Supplemental Procedures Quality Assessment of Template RNA 52 This section gives a general guideline for template RNA quality assessment before proceeding with amplification or hybridization Although optional this step is highly recommended High quality RNA have minimal residual protein gDNA or organic solvent from isolation As such A260 A280 and A260 230 ratios are above 2 0 or 1 8 respectfully Genomic DNA is removed with a high quality DNase treatment Make sure you determine the integrity of the input template RNA before you label amplify and hybridize respectively Use the Agilent 2100 bioanalyzer The RNA 6000 Nano LabChip kit can be used to analyze total RNA mRNA or cRNA with the appropriate assay at the assay specified concentration For low concentration samples consider using the RNA 6000 Pico LabChip kit For the assessment of total RNA quality the Agilent 2100 Expert Software automaticall
20. ate Browser and select Online Update You can also download the latest grid templates from Agilent web site at http earray chem agilent com After downloading you must add the grid templates to the Grid Template Browser After a new grid template is added to the Grid Template Browser remember to specify the default protocol for the new grid template if you want the Feature Extraction program to automatically assign a FE protocol to an extraction set Verify that the correct protocol is assigned to each extraction set in the Protocol Name column To assign a different protocol to an extraction set select from the pull down menu The appropriate protocol begins with GE1 for one color analysis The protocols automatically distinguish the formats for processing the data If a protocol is not available to select from the pull down menu you must import it to the FE Protocol Browser To import right click FE Protocol Browser select Import Browse for the FE protocol xml and click Open to load the protocol into the FE database Visit the Agilent web site at www agilent com chem feprotocols to download the latest protocols These FE Protocols were optimized using data from Agilent catalog arrays which have many replicated probes and validated Negative Control probes If custom arrays without enough replicated probes are used or arrays with custom probes designated as Negative Control probes are used the default FE Protocols may not b
21. c and flammable and must be used in a suitable fume hood This solution contains acetonitrile and must be disposed of in a manner consistent with disposal of like solvents Gloves and eye face protection should be used during every step of this protocol especially when handling acetonitrile and the Stabilization and Drying Solution One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 1 Before You Begin Agilent Oligo Microarrays For more information on microarray designs visit the following web site http www chem agilent com To get design files or create a custom design go to the Agilent eArray web site at http earray chem agilent com Store entire kit at room temperature After breaking foil on microarray pouch store microarray slides at room temperature in the dark under a vacuum dessicator or nitrogen purge box Do not store microarray slides in open air after breaking foil Eight microarrays printed on each 1 inch x 3 inch glass slide Catalog SurePrint HD Microarray Table 1 Catalog SurePrint HD Microarrays Part Number Description G4813A 020097 E coli Gene Expression Microarray 8x15K 1 slide Custom Microarrays Table2 Custom SurePrint HD Microarrays Part Number Description G2509F Custom Gene Expression Microarray 8x 15K Table3 Custom SurePrint G3 Microarrays Part Number Description G4102A SurePrint G3 Custom Gene Expression Microarray 8x60K 10 One C
22. d on select non Agilent scanners Please see Feature Extraction Compatibility Matrix for Non Agilent scanners for scanner compatibility and settings http www chem agilent com Library usermanuals Public G1662 90043 Sc annerCompatibilityMatrix pdf Agilent can guarantee the quality of data only if the data comes from Agilent microarrays scanned on Agilent scanners To get scanner profiles from Agilent For Scan Control 9 1 3 or later go to http www genomics agilent com article jsp pageId 2610 For Scan Control 8 x go to http www genomics agilent com article jsp pageId 2074 Agilent SureScan Microarray Scanner 1 Put assembled slide holders into the scanner cassette 2 Select the protocol AgilentHD GX 1color AgilentHD GX 1color for HD format 3 Verify that the Scanner status in the main window says Scanner Ready 4 Click Start Scan Agilent C Scanner Settings 1 Put assembled slide holders with or without the ozone barrier slide cover into scanner carousel 2 Select Start Slot m End Slot where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located 3 Select Profile AgilentHD GX 1color 4 Verify scan settings for one color scans See Table 14 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 41 2 Procedures CAUTION Do not scan G3 microarrays with HD format settings The resolution of the res
23. de is called the active side The numeric barcode is on the inactive side of the slide CAUTION You must familiarize yourself with the assembly and disassembly instructions for use with the Agilent Microarray Hybridization Chamber G2534A and gasket slides Practice slide kits are available In this processing and hybridization procedure the hybridization mixture is applied directly to the gasket slide and not to the active side of the oligo microarray Instead the active side of the oligo microarray is put on top of the gasket slide to form a sandwich slide pair To avoid damaging the microarray always handle glass slides carefully by their edges Wear powder free gloves Never touch the surfaces of the slides If you do you may cause irreparable damage to the microarray Never allow the microarray surface to dry out during the hybridization process and washing steps One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 67 4 68 General Microarray Layout and Orientation Agilent oligo microarray 1 microarray slide format as imaged on the Agilent microarray scanner Microarrays are printed on the side of the glass labeled with the Agilent bar code also referenced as active side or front side 68 91100 LL FILI TTE IT JA Agilent Microarray Agilent microarray slide holder for Scanner B Scanner Scans and C left or SureScan microarray scan
24. e In general follow Biosafety Level 1 BL1 safety rules Refer to the Fairplay III Microarray Labeling Kit User Guide for more procedural notes 8 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Before You Begin Safety Notes CAUTION im 1 Inspect the Stabilization and Drying Solution bottle for chips or cracks prior to use Failure to do so may result in bottle breakage Wear appropriate personal protective equipment PPE when working in the laboratory Cyanine dye reagents are potential carcinogens Avoid inhalation swallowing or contact with skin LiCl is toxic and a potential teratogen May cause harm to breastfed babies Possible risk of impaired fertility Harmful if inhaled swallowed or contacts skin Target organ central nervous system Wear suitable PPE LiCl is a component of the 2x Hi RPM Hybridization Buffer Lithium dodecyl sulfate LDS is harmful by inhalation and irritating to eyes respiratory system and skin Wear suitable PPE LDS is a component of the 2x Hi RPM Hybridization Buffer Triton is harmful if swallowed Risk of serious damage to eyes Wear suitable PPE Triton is a component of the 2x Hi RPM Hybridization Buffer and is an additive in wash buffers Acetonitrile is a flammable liquid and vapor Harmful if inhaled swallowed or contacts skin Target organs liver kidneys cardiovascular system and CNS Stabilization and Drying Solution is toxi
25. e Extraction Software Reference Guide You can download this guide from the Agilent web site at www agilent com chem dnamanuals protocols One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol One Color Microarray Based Prokaryote Analysis Protocol 4 Reference Kit Contents 64 Supplemental User Guides 66 Microarray Handling Tips 67 General Microarray Layout and Orientation 68 Array Sample tracking microarray slides 71 This chapter contains reference information related to the protocol and Feature Extraction default parameter settings GE Agilent Technologies 63 4 64 Reference Kit Contents The content of the kits used in this protocol required and optional are listed here Table 19 RNA Spike In Kit One Color Content Spike Mix Dilution Buffer Table 20 FairPlay III Microarray Labeling Kit Content 10x AffinityScript Reaction Buffer clear cap Oligo d T primer pink cap 0 1 M DTT green cap DEPC water green cap 20x dNTP Mix with amino allyl dUTP green cap RNase Block purple cap 2x Coupling Buffer clear cap Glycogen green cap DMSO green cap AffinityScript HC Reverse Transcriptase purple cap Random Primers pink cap DNA binding solution Microspin cup Receptacle tube 2 mL One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Reference 4 Table 21 Gene Expression Hybridization Kit Content
26. e optimal When the Agilent XDR scanned images are added to Feature Extraction software version 9 1 or later the High and Low images are automatically combined for data extraction Images are not combined with non Agilent scanned images 20 bit single images from the C Scanner are equivalent to 16 bit XDR images from the B Scanner One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 5 Save the FE Project fep by selecting File gt Save As and browse for desired location 6 Verify that the icons for the image files in the FE Project Window no longer have a red X through them A red X through the icon indicates that an extraction protocol was not selected If needed reselect the extraction protocol for that image file 7 Select Project gt Start Extracting 8 After the extraction is completed successfully view the QC report for each extraction set by double clicking the QC Report link in the Summary Report tab Determine whether the grid has been properly placed by inspecting Spot Finding at the Four Corners of the Array See Figure 7 If a QC Metric Set has been assigned to the FE Project you can view the results of the metric evaluation in three ways Project Run Summary includes a summary sentence QC Report includes both a summary on the header and a table of metric values QC Chart includes a view of the values of each metric compared across all extractions in FE P
27. ed Prokaryote Analysis FairPlay Ill Labeling Protocol 53 3 Supplemental Procedures Step 2 Assess the quality using the Agilent 2100 Bioanalyzer 1 Choose the kit and assay according to your needs Typically the RNA Nano 6000 kit and assay will be appropriate Ensure the 2100 bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Start the Agilent 2100 Expert program version B 02 06 or higher turn on the 2100 bioanalyzer and check communication 4 Prepare the chip samples and ladder as instructed in the reagent kit guide Load the prepared chip into the 2100 bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results Template RNA results total RNA The resulting electropherogram should have at least two distinct peaks representing the prokaryotic 16S and 23S ribosomal RNA Additional bands are the lower marker and the potentially 5S RNA Presence of 5S RNA depends on the purification method generally showing lower abundance in column purified total RNA see Figure 8 Degradation of RNA samples can lead to compromised array results Both the electropherogram and the RIN values can help determine the quality of the sample 54 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Prot
28. er Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indi cated conditions are fully under stood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol In this Guide This document describes the Agilent recommended procedures for the preparation and labeling of complex biological targets and hybridization washing scanning and feature extraction of Agilent 60 mer oligonucleotide microarrays for microarray based one color gene expression analysis 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start an experiment 2 Procedures This chapter describes the steps to prepare samples with the Fairplay III Microarray Labeling Kit hybridize wash and scan gene expression microarrays and to extrac
29. erged in Gene Expression Wash Buffer 1 pry the sandwich open from the barcode end only a Slip one of the blunt ends of the forceps between the slides b Gently turn the forceps upwards or downwards to separate the slides c Let the gasket slide drop to the bottom of the staining dish d Remove the microarray slide and put into slide rack in the slide staining dish 2 containing Gene Expression Wash Buffer 1 at room temperature Minimize exposure of the slide to air Touch only the barcode portion of the microarray slide or its edges 9 Repeat step 6 through step 8 for up to seven additional slides in the group A maximum of eight disassembly procedures yielding eight microarray slides is advised at one time in order to facilitate uniform washing 10 When all slides in the group are put into the slide rack in slide staining dish 312 stir using setting 4 for 1 minute 11 During this wash step remove Gene Expression Wash Buffer 2 from the 37 C water bath and pour into the Wash 2 dish One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 59 3 60 Supplemental Procedures 12 Transfer slide rack to slide staining dish 3 containing Gene Expression Wash Buffer 2 at elevated temperature Stir using setting 4 for 1 minute 13 Remove the slide rack from Gene Expression Wash Buffer 2 and tilt the rack slightly to minimize wash buffer carry over Immediately transfer the slide rack to slide staining dish 4 con
30. idization An instructional video that shows hybridization and washing steps can be found at http genomics agilent com Search for Running a microarray experiment If you are a first time user practice the hybridization process before you begin Use water instead of blocking mix and use a clean microscope slide and a gasket slide Make sure you mix and apply the hybridization solution with minimal bubbles Practice the hyb assembly and the slide disassembly and wash CAUTION You must calibrate the hybridization oven regularly for accuracy of the collected data Refer to Agilent G2545A Hybridization Calibration Procedure p n G2545 90002 version A1 or higher for more information Step 1 Prepare the 10x Blocking Agent 1 Add 500 uL of nuclease free water to the vial containing lyophilized 10x Gene Expression Blocking Agent supplied with the Gene Expression Hybridization Kit or add 1250 uL of nuclease free water to the vial containing lyophilized large volume 10x Gene Expression Blocking Agent 2 Gently mix on a vortex mixer If the pellet does not go into solution completely heat the mix for 4 to 5 minutes at 37 C 3 Drive down any material that sticks to the tube walls or cap by spinning in a centrifuge for 5 to 10 seconds Divide the 10x Gene Expression Blocking Agent into aliquots small enough to keep the freeze thaw cycle to 5 times or less Store at 20 C for up to two months Before use repeat step 2 and step 3
31. indicate the date of addition Triton X 102 10 can be added to smaller volumes of wash buffer as long as the final dilution of the 10 Triton X 102 is 0 005 in the Gene Expression wash buffer solution 34 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 2 Prewarm Gene Expression Wash Buffer 2 Warm the Gene Expression Wash Buffer 2 to 37 C as follows 1 Dispense 1000 mL of Gene Expression Wash Buffer 2 directly into a sterile storage bottle Repeat until you have enough prewarmed Wash Buffer 2 solution for your experiment 2 Tightly cap the sterile storage bottle and put in a 37 C water bath the night before washing arrays Alternatively remove the plastic cubitainer from the box and put it in a 37 C water bath the night before washing the arrays Step 3 Prepare the equipment Always use clean equipment when doing the hybridization and wash steps Designate and dedicate dishes to one color experiments Solvent wash Wash staining dishes racks and stir bars with acetonitrile or isopropyl alcohol to avoid wash artifacts on your slides and images Use acetonitrile for equipment that was exposed to Stabilization and Drying Solution Use isopropyl alcohol for equipment that was not exposed to Stabilization and Drying Solution WARNING Conduct solvent washes in a vented fume hood 1 Add the slide rack and stir bar to the staining dish 2 Transfer the staining dish
32. inds to the silica based fiber matrix seated inside the microspin cup Washing steps remove buffer salts and uncoupled fluorescent dye from the bound cDNA The cDNA is eluted from the matrix using a low ionic strength solution Do not use the DNA binding solution and microspin cups provided in the FairPlay III Microarray Labeling Kit in conjunction with alternative purification protocols 1 Prepare microspin cup elution buffer a Add 1 M HCl to 10 mM Tris base until the pH reaches 8 5 b Ifthe elution buffer is made by diluting a higher molarity Tris base pH 8 5 to a final molarity of 10 mM verify that the pH is still 8 5 If not adjust the pH to 8 5 using either HCl or NaOH 2 Prepare 80 Sulfolane a Incubate the 100 Sulfolane in a 37 C water bath until liquefied 100 sulfolane is a solid at room temperature 80 Sulfolane solution is a liquid at room temperature and can be stored at room temperature for at least a month b Add 1 mL of DNase RNase free distilled water to 4 mL of 100 Sulfolane to make 5 mL of 80 sulfolane 3 Add 90 uL of DNase RNase free distilled water to the 10 uL labeled cDNA 4 Combine 100 uL of DNA binding solution and 100 uL of 80 Sulfolane mixture Mix well on a vortex mixer The two solutions must be well mixed before use 5 Add the 200 uL of DNA binding solution and 80 Sulfolane mixture to the labeled cDNA and mix on a vortex mixer 6 Puta microspin cup into a 2 mL receptacle tube One
33. is of Agilent microarray data Use GeneSpring GX 11 5 or later Note that the default normalization scheme for Agilent one color data in the GeneSpring GX 11 5 or later program is 75th percentile scaling For more information on the GeneSpring GX program go to http www agilent com chem genespring One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 61 3 62 Supplemental Procedures To use Feature Extraction To normalize Agilent one color microarray data without the GeneSpring program use the 75th percentile value for each microarray assay in the Agilent Feature Extraction text file 1 Generate a Feature Extraction text file 2 Find the STATS Table in the middle section of the text file This section describes the results from the array wide statistical calculations 3 Find the 75th percentile value of the non control signals under the column with the heading gPercentileIntensityProcessedSignal 4 Divide each of the green processed signals gProcessedSignal by the 75th percentile signal gPercentileIntensityProcessedSignal to generate the 75th percentile normalized microarray processed signals You can further scale the resulting 75th percentile normalized signals by a constant such as the average of the 75th percentile signals of the arrays in the experiment For more information on the output from the Agilent Feature Extraction program please refer to the Agilent G2567AA Featur
34. ithout notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com put
35. n FE program To get the most recent Feature Extraction protocols for gene expression go to www agilent com chem feprotocols 2 Add the images tif to be extracted to the FE Project a Click Add New Extraction Set s icon on the toolbar or right click the Project Explorer and select Add Extraction b Browse to the location of the tif files select the tif file s and click Open To select multiple files use the Shift or Ctrl key when selecting The FE program automatically assigns a default grid template and protocol for each extraction set if the following conditions are met For auto assignment of the grid template the image must be generated from a Agilent scanner and have an Agilent barcode For auto assignment of the One Color Gene Expression FE protocol the default Gene Expression protocol must be specified in the FE Grid Template properties To access the FE Grid Template properties double click on the grid template in the Grid Template Browser 44 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 3 Set FE Project Properties a Select the Project Properties tab b In the General section enter your name in the Operator text box c In the Input section verify that at least the following default settings as shown in Figure 5 are selected d In the Other section choose a QC Metric Set for the project For Agilent one color microarrays select GE1 QCMT Sep09 Fo
36. nd ensure that the baseline is always flat at 0 which is indicated by a thick black horizontal line If the baseline deviates from 0 and is no longer a flat horizontal line reblank the instrument with 10 mM Tris base pH 8 5 then remeasure the sample Print the results If printing the results is not possible record the following values cyanine 3 dye concentration pmol uL DNA absorbance ratio 260 nm 280 nm cDNA concentration ng uL One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 21 2 Procedures 10 Determine the yield and specific activity of each reaction as follows a Use the concentration of cDNA ng uL to determine the ug cDNA yield as follows Concentration of cDNA x 50 uL elution volume 1000 b Use the concentrations of cDNA ng uL and cyanine 3 pmol uL to determine the specific activity as follows ug of cDNA Concentration of Cy3 pouncenmaten GERDNA ee DECUS GDNA 11 Examine the yield and specific activity results CAUTION If the yield is 650 ng or 13 ng L and the specific activity is 40 pmol of Cy3 per ug of cDNA do not proceed to the hybridization step Repeat cDNA preparation If labeling results are poor please refer to Quality Assessment of Template RNA on page 52 for general guidance and procedural recommendations on quality assessment of RNA 28 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Hybr
37. ner through the glass Back side scanning right Figure 9 Agilent microarray slide and slide holder The opposite or non active numerically barcoded side is shown Agilent oligo microarray formats and the resulting microarray design files are based on how the Agilent microarray scanner images 1 inch x 3 inch glass slides Agilent designed its microarray scanner to scan through the glass slide back side scanning The glass slide is securely placed in an Agilent microarray slide holder with the Agilent labeled barcode facing the opening of the slide holder on SureScan Microarray Scanner G4900DA or facing the inside of the slide holder C scanner G2565CA In this orientation the active side containing the microarrays is protected from potential damage by fingerprints and other elements Once securely placed the numeric barcode non active side of the slide is visible from the outside of the slide holder Figure 9 depicts how the Agilent microarray scanner reads the microarrays and how this relates to the microarray design files that Agilent generates during the manufacturing process of its in situ synthesized oligonucleotide microarrays Thus if you have a scanner that reads microarrays from the front side of the glass slide the collection of microarray data points will be different in relation to the microarray design files supplied with the Agilent oligo microarray kit you purchased The
38. o Reactive Dye is unopened Bring tube to room temperature before you open it Resuspend in 45 uL DMSO green cap Use the high purity DMSO green cap provided in the kit Do not substitute another DMSO The unused dye can be stored in single use aliquots and stored at 20 C in the dark for several months Mix gently to ensure that the pellet is completely soluble DMSO green cap is hygroscopic and will absorb moisture from the air Water absorbed from the air will react with the NHS ester portion of the dye and significantly reduce or eliminate dye cDNA coupling efficiency To reduce absorption allow the dye to reach room temperature before opening and store the DMSO green cap at room temperature Do not leave either the dye or DMSO green cap uncapped when not in use During storage tightly cap the resuspended dye and store at 20 C in the dark Add 5 uL of Cy3 Mono Reactive Dye to the cDNA If the dye was stored at 20 C before use allow the dye to reach room temperature before you open the container Mix by gently pipetting up and down Incubate for 30 minutes at room temperature in the dark One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 5 Purify the dye coupled cDNA Use the reagents in the FairPlay III Microarray Labeling Kit In summary In the presence of a chaotropic salt introduced by the DNA binding solution included in this kit the dye coupled cDNA b
39. ocol Supplemental Procedures 3 120 23S l 25 200 500 1000 2000 4000 Figure 8 Analysis of Escherichia coli total RNA with the Prokaryote Total RNA Nano assay Ribosomal RNA peaks are clearly defined and indicated as 16S and 238 This high quality RNA sample has a RIN value of 10 For general assistance on evaluation of total RNA with emphasis on the RNA integrity number see the corresponding application note RNA integrity number RIN Standardization of RNA quality control 5989 1165EN To download application notes regarding the 2100 bioanalyzer visit Agilent web site at www agilent com chem labonachip One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 55 3 Supplemental Procedures Preventing Ozone Related Problems 56 The Agilent one color platform is robust in environments where the ozone level is 50 ppb approximately 100 ug m or less Beyond this level ozone can significantly affect cyanine 3 signal and compromise microarray performance For Scanner C and Scanner B the Agilent Ozone Barrier Slide cover is designed to protect against ozone induced degradation of cyanine dyes and is recommended when using Agilent oligo based microarrays in high ozone environments See step 14 on page 39 For the Agilent SureScan scanner two built in mechanisms minimize dye signal degradation by ozone and other dye oxidants SureScan slide holder with an integrated ozone barrier in its lid
40. olor Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Required Equipment Table 5 Required Equipment Before You Begin 1 Description Vendor and part number Agilent Microarray Scanner Hybridization Chamber stainless Hybridization gasket slides 8 microarrays slide 5 slides box Go to www agilent com genomics to see all available kit configurations Hybridization oven temperature set at 65 C Hybridization oven rotator for Agilent Microarray Hybridization Chambers nuclease free 1 5 mL microfuge tube magnetic stir bar x2 magnetic stir plate x2 circulating water baths or heat blocks set to 37 C 40 C 60 C 65 C 70 C and 80 C microcentrifuge sterile storage bottle spectrophotometer micropipettor slide staining dish with slide rack x3 vacuum concentrator clean forceps ice bucket powder free gloves sterile nuclease free aerosol barrier pipette tips vortex mixer Agilent p n G4900DA G2565CA or G2565BA Agilent p n G2534A Agilent p n G2534 60014 Agilent p n G2545A Agilent p n G2530 60029 Ambion p n 12400 or equivalent Corning p n 401435 or equivalent Corning p n 6795 410 or equivalent Corning p n 6795 420 or equivalent Eppendorf p n 5417R or equivalent Nalgene 455 1000 or equivalent NanoDrop p n ND 1000 UV VIS or equivalent Pipetman P 10 P 20 P 200 P 1000 or equivalent Thermo Shandon p n 121 or equivalent Thermo Scientific p n DNA 120 115
41. on with pH 7 9 when no less than 50 uL of elution buffer is added directly onto the fiber matrix at the bottom of the microspin cup and when the tube is incubated for 5 minutes c Snap the cap of the nuclease free 1 5 mL microfuge tube onto the microspin cup and spin the tube in a microcentrifuge at maximum speed for 30 seconds d Open the lid of the microcentrifuge tube and recover the flow through that contains the purified labeled cDNA 18 Elute additional labeled cDNA a Elute additional labeled cDNA by pipetting the flow through back onto the fiber matrix of the same microspin cup b Re seatthe spin cup on the same 2 mL receptacle tube that contained the liquid from the first pass elution c Incubate the tube at room temperature for 5 minutes Snap the cap of the nuclease free 1 5 mL microfuge tube onto the microspin cup and spin the tube in a microcentrifuge at maximum speed for 30 seconds e Open the lid of the microcentrifuge tube and recover the flow through containing the purified labeled cDNA 19 Repeat step 18 to harvest one final elution from the microspin cup One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 25 2 26 Procedures 20 Remove a 2 uL sample of the labeled cDNA for analysis of dye incorporation using a small volume spectrophotometer such as a NanoDrop instrument See Step 6 Quantify the cDNA on page 27 21 If needed reduce the volume of the labeled cDNA
42. or equivalent One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 11 1 12 Before You Begin Table 5 Required Equipment continued Description Vendor and part number timer nitrogen purge box for slide storage Required Reagents Table 6 Required Reagents Description Vendor and part or catalog number RNA Spike In Kit One Color Gene Expression Hybridization Kit Gene Expression Wash Buffer Kit FairPlay IIl Microarray Labeling Kit 10 reactions 30 reactions Cy3 Mono Reactive Dye DNase RNase free distilled water RNeasy Mini Kit Sulfolane ethanol 95 to 100 molecular biology grade Milli Q water or equivalent DNA size standard useful range 400 1000 bp 1M NaOH 1M HCI 10 mM Tris base adjust pH to 8 5 with HCI 3 M Sodium Acetate pH 4 5 isopropyl alcohol molecular biology grade Agilent p n 5188 5282 Agilent p n 5188 5242 Agilent p n 5188 5327 Agilent p n 252009 Agilent p n 252012 GE Healthcare p n PA23001 Invitrogen p n 10977 015 Qiagen p n 74104 Sigma Aldrich p n 122209 Sigma Aldrich p n E7023 6x500ML One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Before You Begin 1 Optional Equipment Reagents Table 7 Optional Equipment Reagents Description Vendor and part number 2100 Bioanalyzer Agilent p n G2939AA RNA 6000 Nano Assay Kit RNA Series II Kit Agilent p n 5067 1511 Stabilization and D
43. pment 35 Step 4 Wash the microarray slides 37 Scanning and Feature Extraction 41 Step 1 Scan the slides 41 Step 2 Extract data using Agilent Feature Extraction Software 44 The Agilent One Color Microarray based Model Organism Analysis uses cyanine 3 labeled targets to measure gene expression in experimental and control samples Figure 1 is a standard workflow for sample preparation and array hybridization design RE Agilent Technologies 15 2 Procedures Figure 1 Workflow for sample preparation and array processing 16 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 dNTP mix with amino allyl dUTP dNTP mix with Reverse transcription with amino allyl dUTP AffinityScript HC RT at 42 C to make first strand cDNA C Evenly labeled cDNA Figure 2 The FairPlay IIl Microarray Labeling Kit creates evenly labeled cDNA The FairPlay kit labels sample with fluorescent dyes using a chemical coupling method First strand synthesis incorporates an amino allyl dUTP This reactive group is then coupled to a conjugated dye resulting in evenly labeled cDNA When you generate targets for a one color microarray experiment only the Cy3 labeled sample is produced and hybridized One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 17 2 Procedures Sample Preparation Step 1 Dilute Spike in Solution Equilibrate a water bath to 37 C Thaw Spike Mix concentra
44. r outputs that can be imported into Rosetta Resolver select MAGE and JPEG B General Operator Unknown El Input Number of Extraction Sets Included 0 Output and Data Transfer E Outputs MAGE None JPEG None B TEXT Local file only Visual Results Local file only Grid Local file only QC Report Local file only FTP Send Tiff File False EJ Local File Folder Same As Image True R esu ts Fo der FTP Setting E Automatic Protocol Assignment Highest Priority Default Protocol Grid Template Default Project Default Protocol Automatic Grid Template Assignment Use Grid file if available True B Other QC Metric Set External DyeNorm List File Overwrite Previous Results False Figure 5 Default settings 4 Checkthe Extraction Set Configuration a Select the Extraction Set Configuration tab b Verify that the correct grid template is assigned to each extraction set in the Grid Name column To assign a different grid template to an extraction set select one from the pull down menu One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 45 2 46 Procedures If a grid template is not available to select from the pull down menu you must add it to the Grid Template Browser To add right click inside the Grid Template Browser select Add Browse for the design file xml and click Open to load grid template into the FE database To update to the latest grid templates via Online Update right click Grid Templ
45. refore please take a moment to become familiar with the microarray layouts for each of the Agilent oligo microarrays and the layout information as it pertains to scanning using a front side scanner One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Reference 4 Non Agilent front side microarray scanners When imaging Agilent oligo microarray slides you must determine Ifthe scanner images microarrays by reading them on the front side of the glass slide Agilent labeled barcode side of the slide and If the image produced by the non Agilent scanner is oriented in a portrait or landscape mode Agilent labeled barcode left side right side up or down as viewed as an image in the imaging software see Figure 10 This changes the feature numbering and location as it relates to the microarray design files found on the CD in each Agilent oligo microarray kit Microarray layout maps are available from Agilent For more information go to www agilent com chem dnamanuals protocols and download Agilent Microarray Formats Technical Drawings with Tolerance publication G4502 90001 This document contains visual references and guides that will help you determine the feature numbering as it pertains to your particular scanner configuration One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 69 70 4 Reference Front side ar code up portrait
46. removal of the ethanol before proceeding to the dye coupling reaction Add 4 uL of 3 M Sodium Acetate pH 4 5 to the reaction Add 1 uL of 20 mg mL Glycogen green cap to the reaction Add 100 uL of ice cold 95 ethanol Incubate at 20 C for at least 30 minutes The reaction can be stored at this point for several days or up to 2 months 5 Spin the reaction at 13 000 to 14 000 x g for 15 minutes at 4 C Carefully remove the supernatant 6 Wash the pellet with 0 5 mL ice cold 70 ethanol and spin at 13 000 to 14 000 x g for 15 minutes at 4 C Carefully remove the supernatant and allow the pellet to air dry Aa Q N A vacuum dryer can be used but the pellet must not be excessively dried One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 21 2 Procedures Step 4 Label the modified cDNA CAUTION Note that dye packs can be stored at 4 C until use but make sure they are at room temperature before you open and add DMSO Do not scale down the dye coupling instruction If you do the reaction with reduced volumes significantly reduced coupling efficiency can result 22 4 Resuspend cDNA pellet in 5 uL of 2x Coupling Buffer clear cap Excessively dried pellets are difficult to get back into solution Gently heat at 37 C for 15 minutes to make sure that the precipitate is soluble before use A visible precipitate may be seen in the 2x Coupling Buffer clear cap If the tube of Cy3 Mon
47. rier Slide Cover described in this topic SureScan microarray scanner uses a slide holder with a built in ozone barrier When setting up the apparatus for the washes be sure to do so near the water bath containing the pre warmed Wash 2 solutions Table 13 lists the wash conditions for the wash procedure Table 13 Wash conditions Dish Wash Buffer Temperature Time Disassembly 1 Gene Expression Wash Buffer 1 Room temperature 1st wash 2 Gene Expression Wash Buffer 1 Room temperature 1 minute 2nd wash 3 Gene Expression Wash Buffer2 Elevated temperature 1 minute The elevated temperature of the second wash step is usually around 31 C due to cooling by the room temperature dish and the rack of arrays 1 Completely fill slide staining dish 1 with Gene Expression Wash Buffer 1 at room temperature 2 Puta slide rack into slide staining dish 2 Add a magnetic stir bar Fill slide staining dish 2 with enough Gene Expression Wash Buffer 1 at room temperature to cover the slide rack Put this dish on a magnetic stir plate 3 Put the empty dish 3 on the stir plate and add a magnetic stir bar Do not add the prewarmed 37 C Gene Expression Wash Buffer 2 until the first wash step has begun 4 Remove one hybridization chamber from incubator and record time Record whether bubbles formed during hybridization and if all bubbles are rotating freely One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 3
48. roject Refer to the application note Enhanced Quality Assessment Using Agilent Feature Extraction QC Metric Sets Thresholds and Charting Tools p n 5989 5952EN for more details on quality assessment and troubleshooting with the Feature Extraction QC Report This technical note can be downloaded from the Agilent web site at www agilent com Search for the part number 5989 5952EN One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 47 2 48 Procedures Step 2 Extract data using Agilent Feature Extraction Software Automatic Download from eArray Feature Extraction version 10 7 or higher can automatically download Grid Templates protocols and QC metrics QCM or QCMT To set this up in the eArray Login Setting dialog box under Advanced Options click Use eArray server during extraction See Figure 6 eArray Login Setting x eArray Login Setting earray chem agient com User Name Password m Test Connection For an eArray account please register first Register d Option ACV SNCS Opuons I Use eArray server during extraction Check for updates of grid template c On starting FE check for protocol update from eArray server Don t show this dialog again 1 Figure 6 eArray Login Setting One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 QC Report Agilent Technologies 1 Color Gene Expression Date Image Protocol User Name
49. rying Solution Agilent p n 5185 5979 Ozone Barrier Slide Cover Agilent p n G2505 60550 slide box Corning p n 07201629 acetonitrile Sigma p n 271004 1L Recommended when processing microarrays in high ozone environment Required Hardware and Software Table 8 Description Feature Extraction software 10 7 1 or later Agilent Scan Control software Refer to Agilent Scanner user guide for specifications For system and supported Internet Explorer Adobe Reader versions please see the System Requirements for your Feature Extraction and Scan Control Software Optional Software Table 9 Description GeneSpring GX 11 5 or later One Color Microarray Based Prokaryote Analysis FairPlay III Labeling Protocol 13 14 This page intentionally left blank One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol One Color Microarray Based Prokaryote Analysis Protocol 2 Procedures Sample Preparation 18 Step 1 Dilute Spike in Solution 18 Step 2 Synthesize cDNA 19 Step 3 Purify cDNA 21 Step 4 Label the modified cDNA 22 Step 5 Purify the dye coupled cDNA 23 Step 6 Quantify the cDNA 27 Hybridization 29 Step 1 Prepare the 10x Blocking Agent 29 Step 2 Prepare hybridization samples 30 Step 3 Prepare the hybridization assembly 31 Microarray Wash 34 Step 1 Add Triton X 102 to Gene Expression wash buffers 34 Step 2 Prewarm Gene Expression Wash Buffer 2 35 Step 3 Prepare the equi
50. t data using the Agilent Feature Extraction Software 3 Supplemental Procedures This chapter contains instructions for quality assessment of template RNA and labeled cDNA and steps to prevent ozone related problems 4 Reference This chapter contains reference information related to the protocol One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 3 What s new in 2 0 What s new in 1 4 What s in 1 3 Added solvent wash for glassware to prepare for microarray wash Added list of supported microarrays Added note to calibrate hybridization oven on a regular basis for accuracy of the collected data Updated loading instructions for hybridization oven Added reference to compatibility matrix for non Agilent scanners Expanded instructions to prepare hybridization assembly Updated product labeling statement Support for Agilent SureScan microarray scanner Change of formula to quantify cDNA before hybridization This protocol is the one color version of the Two Color Microarray Based Prokaryote Analysis FairPlay III Labeling Protocol p n G4813 90010 4 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Content 1 Before You Begin 7 Procedural Notes 8 Safety Notes 9 Agilent Oligo Microarrays 10 Required Equipment 11 Required Reagents 12 Optional Equipment Reagents 13 Required Hardware and Software 13 Optional Software 13 2 Procedures
51. taining acetonitrile and stir using setting 4 for less than 10 seconds 14 Transfer the slide rack to dish 5 filled with Stabilization and Drying Solution and stir using setting 4 for 30 seconds 15 Slowly remove the slide rack trying to minimize droplets on the slides It should take 5 to 10 seconds to remove the slide rack 16 Discard used Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 17 Repeat steps 1 through 16 for the next group of eight slides using fresh Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 pre warmed to 37 C 18 Scan slides immediately to minimize the impact of environmental oxidants on signal intensities If necessary store slides in orange slide boxes in a nitrogen purge box in the dark One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Supplemental Procedures 3 Normalizing Agilent One Color Microarray Data When comparing data across a set of one color microarrays a simple linear scaling of the data is usually sufficient for most experimental applications Agilent has determined that the signal value of the 75th percentile of all of non control probes on the microarray is a more robust and representative value of the overall microarray signal as compared to the median or 50th percentile signal Therefore use the 75th percentile signal value to normalize Agilent one color microarray signals for inter array comparisons To do downstream analys
52. te and mix vigorously in a vortex mixer Heat at 37 C in a circulating water bath for 5 minutes Mix the spike mix vigorously on a vortex mixer Briefly spin in a centrifuge to spin contents to bottom of tube Add 5 uL of Spike Mix to 15 uL of Dilution buffer This is sufficient for up to 9 labeling reactions ao a Aa c N 18 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Step 2 Synthesize cDNA Use reagents from RNA Spike In Kit One Color FairPlay III Microarray Labeling Kit If you begin with 5 ug of RNA you have enough cDNA for one 8x15K microarray 1 Add 5 ug total RNA to a 1 5 mL microcentrifuge tube in a volume of 10 uL or less If samples are concentrated dilute with water until 5 ug of total RNA is added in at least 2 uL volume with a pipette to ensure the accuracy 2 Add 2 uL of diluted Spike Mix solution to the 5 ug of Total RNA If needed bring the total volume of the Spike Mix RNA solution to 12 uL with nuclease free water 3 Add 1 uL of Random Primers pink cap to the Spike in RNA solution Gently mix on a vortex mixer and briefly spin the tube in a centrifuge 4 Incubate the tube at 70 C for 10 minutes Put reagents on ice and incubate for 5 minutes Immediately prior to use gently mix the components listed in Table 10 for the cDNA Master Mix by adding in the order indicated and put on ice Use a pipette to thoroughly mix the components Make sure the
53. terial away from ignition sources Use gloves and eye face protection in every step of the warming procedures WARNING Failure to follow the outlined process will increase the potential for fire explosion and possible personal injury Agilent assumes no liability or responsibility for damage or injury caused by individuals performing this process 1 Warm the solution slowly in a water bath or a vented conventional oven at 40 C in a closed container with sufficient head space to allow for expansion The original container can be used to warm the solution Container volume is 700 mL and contains 500 mL of liquid If a different container is used maintain or exceed this headspace liquid ratio The time needed to completely redissolve the precipitate is dependent on the amount of precipitate present and may require overnight warming if precipitation is heavy DO NOT FILTER the Stabilization and Drying solution 2 If needed gently mix to obtain a homogeneous solution Mix under a vented fume hood away from open flames or other sources of ignition Warm the solution only in a controlled and contained area that meets local fire code requirements 3 After the precipitate is completely dissolved let the covered solution stand at room temperature allowing it to equilibrate to room temperature and make sure that precipitation does not occur prior to use One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol
54. there are an equal number of empty positions on each of the four rows on the hybridization rack The Gene Expression Wash Buffer 2 needs to be warmed overnight Make sure that you prepare the wash buffer the night before you plan to do the microarray wash See Step 2 Prewarm Gene Expression Wash Buffer 2 One Color Microarray Based Prokaryote Analysis FairPlay III Labeling Protocol 33 2 Procedures Microarray Wash Step 1 Add Triton X 102 to Gene Expression wash buffers This step is optional but highly recommended The addition of 0 005 Triton X 102 10 to the Gene Expression wash buffers reduces the possibility of array wash artifacts Add Triton X 102 10 to Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 when the cubitainer of wash buffer is first opened Do this step to both Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 before use 1 Open the cardboard box with the cubitainer of wash buffer and carefully remove the outer and inner caps from the cubitainer 2 Usea pipette to add 2 mL of the provided Triton X 102 10 into the wash buffer in the cubitainer 3 Replace the original inner and outer caps and mix the buffer carefully but thoroughly by inverting the container 5 to 6 times 4 Carefully remove the outer and inner caps and install the spigot provided with the wash buffer 5 Prominently label the wash buffer box to indicate that Triton X 102 10 has been added and
55. ulting image will not be high enough for data analysis Table 14 C Scanner Scan Settings For HD Microarray Formats Dye channel G green Scan region Agilent HD 61 x 21 6 mm Scan resolution 5 um Tiff file dynamic range 20 bit Green PMT gain 10096 5 Verify that Output Path Browse is set for desired location 6 Verify that the Scanner status in the main window says Scanner Ready 7 Click Scan Slot m n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located Agilent B Scanner Settings Agilent Scan Control software v7 0 01 is required 1 Put slide into slide holder with or without the ozone barrier slide cover with Agilent barcode facing up 2 Putassembled slide holders into scanner carousel 3 Verify scan settings for two color scans See Table 15 For version 7 X to change any settings click Settings gt Modify Default Settings A window pops up from which you can change the settings 42 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 Table 15 B Scanner Scan Settings For All Formats Scan region Scan Area 61 x 21 6 mm Scan resolution um 5 5um scanning mode Single Pass eXtended Dynamic range selected Dye channel Green Green PMT XDR Hi 100 XDR Lo 10 4 Select settings for the automatic file naming Prefi
56. ver the slide rack Put this dish on a magnetic stir plate 3 Put the empty dish 3 on the stir plate and add a magnetic stir bar Do not add the pre warmed 37 C Gene Expression Wash Buffer 2 until the first wash step has begun One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Supplemental Procedures 3 4 Fill slide staining dish 4 approximately three fourths full with acetonitrile Add a magnetic stir bar and put this dish on a magnetic stir plate 5 Fill slide staining dish 5 approximately three fourths full with Stabilization and Drying Solution Add a magnetic stir bar and put this dish on a magnetic stir plate 6 Remove one hybridization chamber from incubator and record time Record whether bubbles formed during hybridization and if all bubbles are rotating freely 7 Prepare the hybridization chamber disassembly a Put the hybridization chamber assembly on a flat surface and loosen the thumbscrew turning counter clockwise b Slide off the clamp assembly and remove the chamber cover c With gloved fingers remove the array gasket sandwich from the chamber base by grabbing the slides from their ends Keep the microarray slide numeric barcode facing up as you quickly transfer the sandwich to slide staining dish 1 d Without letting go of the slides submerge the array gasket sandwich into slide staining dish 1 containing Gene Expression Wash Buffer 1 8 With the sandwich completely subm
57. x1 is set to Instrument Serial Number Prefix2 is set to Array Barcode 5 Verify that the Scanner status in the main window says Scanner Ready Click Scan Slot m n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol 43 2 Procedures Step 2 Extract data using Agilent Feature Extraction Software Feature Extraction is the process by which information from probe features is extracted from microarray scan data allowing researchers to measure gene expression in their experiments To get the most recent Feature Extraction software for gene expression go to the Agilent web site at www agilent com chem fe From the Feature Extraction online Help you can find the Quick Start Guide the detailed User Guide and the Reference Guide The Reference Guide includes descriptions of all feature output and the algorithms used Feature Extraction FE 10 7 1 and later support extraction of one color tif images of Agilent microarrays scanned on Agilent Scanner Images from the Axon Molecular Devices model 4000B can be analyzed with the use of Feature Extraction version 9 5 found at www agilent com chem fe After generating the microarray scan images extract tif images using the Feature Extraction software 1 Open the Agilent Feature Extractio
58. y provides a RNA Integrity Number RIN RIN provides a quantitative value for RNA integrity that facilitates the standardization of quality interpretation Users should define a minimum threshold RIN number based on correlative data in order to eliminate experimental bias due to poor RNA quality Analysis of single stranded RNA provides information on size distribution and concentration It allows relative quantification of fragments within a size range One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Supplemental Procedures 3 Step 1 Prepare for quality assessment Refer to Table 16 and Table 17 to make sure that you have the appropriate analyzer kits and compatible assays Table 16 Analyzer and Kits Description Vendor and part number 2100 Bioanalyzer Agilent p n G2938C or G2939A RNA 6000 Nano LabChip Kit Agilent p n 5067 1511 RNA 6000 Pico LabChip Kit Agilent p n 5067 1513 Table 17 Compatible Assays Description Compatible Assay RNA 6000 Nano LabChip Kit Prokaryotic Total RNA Nano Assay Qualitative range 5 to 500 ng pL RNA 6000 Nano LabChip Kit mRNA Nano Assay Qualitative range 25 to 250 ng uL RNA 6000 Pico LabChip Kit Prokaryotic Total RNA Pico Assay Qualitative range 50 to 5000 pg pyL in water RNA 6000 Pico LabChip Kit mRNA Pico Assay Qualitative range 250 to 5000 pg pL in water The mRNA assays are suitable for analysis of cDNA as well One Color Microarray Bas
59. y slides 8 When all slides in the group are placed into the slide rack in slide staining dish 2 stir using setting 4 for 1 minute 9 During this wash step remove Gene Expression Wash Buffer 2 from the 37 C water bath and pour into the slide staining dish 3 10 Transfer slide rack to slide staining dish 3 that contains Gene Expression Wash Buffer 2 at elevated temperature Stir using setting 4 or a moderate speed setting for 1 minute 11 Slowly remove the slide rack minimizing droplets on the slides It should take 5 to 10 seconds to remove the slide rack If liquid remains on the bottom edge of the slide dab it on a cleaning tissue 12 Discard used Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 One Color Microarray Based Prokaryote Analysis FairPlay Ill Labeling Protocol Procedures 2 13 Repeat step 1 through step 12 for the next group of eight slides using fresh Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 pre warmed to 37 C 14 Put the slides in a slide holder For SureScan microarray scanner Carefully put the end of the slide without the barcode label onto the slide ledge Gently lower the microarray slide into the slide holder Make sure that the active microarray surface with Agilent labeled barcode faces up toward the slide cover Close the plastic slide cover pushing on the tab end until you hear it click For more detailed instruction refer to the Agilent G49

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