miRNA Microarray System with miRNA Complete Labeling Hyb Kit 2.4
Contents
1. To set this up in the eArray Login Setting dialog box under Advanced Options click Use eArray server during extraction See Figure 8 eArray Login Setting X eArray Login Setting earray chem agient com User Name Test Connection For an eArray account please register first Register MOV SNC eS Opuons I Use eArray server during extraction I Check for updates of grid template r I On starting FE check for protocol update from eArray server T cose Figure 8 eArray Login Setting miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 47 2 Procedures Step 2 Extract data using Agilent Feature Extraction Software 48 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 3 Characterization of Total RNA Step 1 Prepare for characterization of total RNA 51 Step 2 Characterize the RNA samples using the Agilent 2100 Bioanalyzer 52 Although this step is optional in the miRNA microarray work flow Agilent strongly recommends that you characterize the total RNA for sample integrity and possible presence of contaminants This chapter gives a general guideline for total RNA and small RNA characterization prior to proceeding with labeling and hybridization Depending on the method used for purification of the total RNA your sample may have contaminants such as phenol salts and or genomic DNA You can2. 4 Add 198 uL of the Dilution Buffer to each of the 10 tubes 5 Add Spike In solution Add 2 uL of the Labeling Spike In solution to each of the 5 tubes that are labeled 1st Dilution Labeling Spike In Add 2 uL of the Hyb Spike In solution to each of the 5 tubes that are labeled 1st Dilution Hyb Spike In tubes 6 Mix well and briefly spin tubes 7 Optional To increase the number of uses of this kit and to prevent excess freeze thaw cycles store aliquots of the spike in solutions in tubes that are appropriately labeled 1st Dilution Labeling Spike In and 1st Dilution Hyb Spike In Store at 80 C 8 Use only one aliquot of the 1st Dilution Labeling Spike In and 1st Dilution Hyb Spike In at a time and store the other aliquots at 80 C miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 17 2 To prepare Spike In solutions Prepare dilutions of the Spike In solution just prior to use Label 4 new tubes 2nd Dilution Labeling Spike In 3rd Dilution Labeling Spike In 2nd Dilution Hyb Spike In 3rd Dilution Hyb Spike In 2 Into each of the 4 tubes add 198 uL of nuclease free water 3 Make the 2nd dilution solutions Add 2 uL from the 1st Dilution Labeling Spike In tube into the 2nd Dilution Labeling Spike In tube Add 2 uL from the Ist Dilution Hyb Spike In tube into the 2nd Dilution Hyb Spike In tube 4 Mix well and spin briefly in a centrifuge Make the 38rd d
3. THEIR AFFILIATES TRUSTEES OFFICERS EMPLOYEES ATTORNEYS AND AGENTS FROM ALL CLAIMS OR DEMANDS MADE AGAINST THEM AND ANY RELATED LOSSES EXPENSES OR COSTS ARISING OUT OF OR RELATING TO YOUR AND YOUR CUSTOMERS USE OF DISPOSITION OF OR CONDUCT REGARDING THE PRODUCT INCLUDING BUT NOT LIMITED TO ANY CLAIMS OF PRODUCT LIABILITY PERSONAL INJURY INCLUDING BUT NOT LIMITED TO DEATH DAMAGE TO PROPERTY OR VIOLATION OF ANY LAWS OR REGULATIONS INCLUDING BUT NOT LIMITED TO CLAIMS OF ACTIVE OR PASSIVE NEGLIGENCE miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol www agilent com In This Book This document describes Agilent s recommended procedures for the preparation and labeling of complex biological targets and hybridization washing scanning and feature extraction of Agilent oligonucleotide microarrays for microarray based miRNA systems Agilent Technologies Inc 2010 2011 Version 2 4 August 2011 Revision A3 G4170 90011 Ee Agilent Technologies
4. 4 Wash the microarray slides The microarray wash procedure for Agilent s miRNA platform must be done in environments where ozone levels are 50 ppb or less For environments in which ozone levels are over 50 ppb use the ozone barrier slide cover 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 10 lists the wash conditions for the wash procedure Table 10 Wash conditions Dish Wash Buffer Temperature Time Disassembly 1 GE Wash Buffer 1 Room temperature 1st wash 2 GE Wash Buffer 1 Room 5 minutes temperature 2nd wash 3 GE Wash Buffer 2 37 C 5 minutes 1 Completely fill slide staining dish 1 with Gene Expression Wash Buffer 1 at room temperature 2 Place a 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 Place this dish on a magnetic stir plate 3 Put the prewarmed 1 5 L glass dish filled with water and containing slide staining dish 3 on a magnetic stir plate with heating element Fill the slide staining dish 3 approximately three fourths full with Gene Expression Wash Buffer 2 warmed to 37 C Add a magnetic stir bar Turn on the heating element and maintain temperature of Gene Expression Wash Buffer 2 at 37 C Use a thermometer to check the temperature 4 Remove one hybridization chamber from the incubator a
5. 4 Scan Control dialog box with default settings for Scanner C e Ifyou use a B scanner with Scan control software version 7 x you need to verify and possibly change the scan settings appropriate for miRNA scans Figure 5 shows the version 7 x Scan Control dialog box Table 12 shows the scan settings appropriate for miRNA scans miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 1 Scan the slides Figure 5 Scan Control dialog box with miRNA setting for Scanner B Table 12 Scan Settings Scan Setting Values for 8x15K 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 5 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 39 2 Procedures CAUTION The scanner settings for miRNA microarray scanning are not the same as the default XDR settings for version 7 x 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 4 Select settings for the automatic file naming Prefix 1 is set to Instrument Serial Number Prefix 2 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
6. Sig Distrib 50 of Sig Distrib 1 of Sig Distrib Negative Control Stats Average Net Signals StdDev Net Signals Average BG Sub Signal p StdDev BG Sub Signal Histogram of Signals Plot 0 1 2 3 Leg of 80 SaS W wistogram of Signals Features NonCtri with BGSubSignal lt 0 18561 Green with the use of Spike In miRNAs Reproducibility CV for Replicated Probes plot 109 SCV br each Replicate Frote Lop _gheciarProcestesSnal SCV for Green Evaluation Metrics for miRNA_QCMT_Sep09 Excellent 4 Good 2 Metric Name Value Excellent Good Evaluate 1sGoodGrid ot NA AddErrorEstimateGreen lt 5 Sto12 AnyColarPrentFeatPopaOL lt 8 Bto1s QNonCtrIMedPrentCVBGSu 4 Otoio 1015 lt 0or gt 15 gTotalSignal7Spctile LabelingSpike InSignal gt 2 50 2 50 HybSpike InSignal gt 2 50 2 50 StringeneySpike InRatio Excellent Good Evaluate miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol E GY 90 120150181 200 261 PT SON S3T oT Mow Median Proc Signal Row Spatial Distribution of Median Signals for each Column NM ae Va ih Example of a QC Report for 8x15K miRNA microarray generated by Feature Extraction version 10 7 3 45 2 46 Procedures Agilent Feature Extraction Result Files Agilent Feature Extraction software generates two text files for each miRNA microarray The text file that uses the name ScannerNum_BarcodeNum_S0 _FEPro
7. and understand before you start an experiment 2 Procedures This chapter describes the steps to prepare Spike In solutions optional samples hybridize wash and scan miRNA microarrays and to extract data using the Feature Extraction Software 3 Characterization of Total RNA This chapter contains instructions for characterization of total RNA 4 Reference This chapter contains reference information related to the protocol and the limited use license miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 3 What s New in 2 4 What s New in 2 3 What s New in 2 2 Support for Agilent SureScan Microarray Scanner Update to list of recommended RNA extraction methods Introduction of SurePrint G3 8x60K microarray format which allows more content to be assayed on a single array without any changes to sample processing The Sureprint G3 8x60K format requires the use of the Agilent C scanner Requirement to use Feature Extraction 10 7 3 with the use of the microRNA Spike In kit Optional column purification during sample preparation Optional use of microRNA Spike In Kit Requirement to use Feature Extraction 10 7 with the use of the microRNA Spike In kit miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Content 1 Before You Begin Procedural Notes 8 Safety Notes 8 Agilent Oligo Microarray Kit Contents 8 Required Equipment 9 Required Reagents 10 Optional Equipment Re
8. by incubating the reaction at 37 C in a circulating water bath or heat block for 30 minutes If needed the samples can be stored at 80 C after incubation A thermocycler in conjunction with PCR plates can be used to process a large number of samples Use a PCR cap to seal the plates Do not use tape miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Procedures 2 Denaturation 1 Add 2 8 uL of 100 DMSO to each sample 2 Incubate samples at 100 C in a circulating water bath or heat block for 5 to 10 minutes Incubate the sample for no less than 5 minutes and no more than 10 minutes or the labeling efficiency of the sample will be affected CAUTION 3 Immediately transfer to ice water bath You must use an ice water bath and not just crushed ice to ensure that the samples remain properly denatured When using a thermocycler transfer the PCR plate to an ice water bath immediately after incubating at 100 C to prevent the RNA from re annealing Thermocyclers do not cool as quickly as is needed 4 Continue to the next step immediately miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 21 2 Procedures CAUTION Ligation 1 Warm the 10X T4 RNA Ligase Buffer at 37 C and vortex until all precipitate is dissolved Make sure that the 10X T4 RNA Ligase Buffer has cooled to room temperature before you proceed Failure to do so will affect the T4 RNA Ligase
9. run Enter sample names and comments in the Data and Assay context 7 Verify the results 52 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol FU 70 60 50 40 30 20 Figure 9 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 25 Characterization of Total RNA RNA 6000 Total RNA Total RNA characterization provides information on the quality of the total RNA which may impact the overall RNA quantification and the microarray miRNA profile The resulting electropherogram typically has at least two distinct peaks representing the 18S and 28S ribosomal RNA The peak representing the lower marker can also be seen as shown in Figure 9 The presence of the 5S RNA peak as shown in Figure 9 depends on the purification method and generally shows lower abundance in column purified total RNA Figure 9 demonstrates the differences in the peaks with varying degrees of total RNA integrity and provides RNA Integrity Number RINs as examples Make sure you define a minimum threshold RIN number based on correlative data in order to eliminate experimental bias due to poor total RNA quality 200 500 1000 2000 4000 nt Analysis of human total RNA with the Eukaryote total RNA Nano assay using three different sam ples with decreasing integrity Red RIN 8 4 Blue RIN 5 9 Green RIN 3 6 Characteristic regions for ribosomal peaks and the lower marker LM are display
10. 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 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 59 4 60 Limited Use License for End Users Nucleotide Analog Component THIS PRODUCT IS SUBJECT TO PROPRIETARY RIGHTS OF GE HEALTHCARE BIO SCIENCES CORP GE HEALTHCARE AND CARNEGIE MELLON UNIVERSITY CMU AND MADE AND SOLD UNDER LICENSE FROM GE HEALTHCARE THE PRODUCT BEARING THIS LABEL IS LICENSED FOR SALE ONLY FOR RESEARCH NOT TO INCLUDE IN VIVO RESEARCH AND IN ADDITION MAY BE USED TO PERFORM RESEARCH NOT TO INCLUDE IN VIVO RESEARCH SERVICES FOR THIRD PARTIES IT IS NOT LICENSED NOR ARE THERE ANY IMPLIED LICENSES FOR ANY OTHER USE INCLUDING WITHOUT LIMITATION COMMERCIAL USES UNLESS EXPRESSLY AUTHORIZED BY GE HEALTHCARE IN WRITING IF YOU INTEND TO USE THIS PRODUCT COMMERCIALLY AND YOU DO NOT HAVE A LICENSE TO USE THIS PRODUCT FOR THE INTENDED COMMERCIAL PURPOSE RETURN THIS PRODUCT UNOPENED TO Agilent Technologies 5301 Stevens Creek Blvd Santa Clara CA 95051 AND ANY MONEY PAID FOR THE PRODUCT WILL BE REFUNDED COMMERCIAL USE shall include 1 sale lease license or other transfer of the material or any material derived or produced from it 2 sale lease license or other grant of rights to use this Material or any material derived or produced from it YOU HEREBY AGREE TO DEFEND INDEMNIFY AND HOLD HARMLESS GE HEALTHCARE AND CMU
11. 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 puter 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 indicated conditions are fully understood 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 2 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol In this Guide This document describes Agilent s recommended procedures for the preparation and labeling of complex biological targets and hybridization washing scanning and feature extraction of Agilent oligonucleotide microarrays for microarray based miRNA systems 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment as well as total RNA extraction recommendations that you should read
12. A Kit Agilent p n G2938C or G2939A Agilent p n 5067 1511 Agilent p n 5067 1513 Agilent p n 5067 1548 Table 14 Recommended Assays Description Compatible Assay RNA 6000 Nano Kit Agilent Eukaryote Total RNA Nano Assay Qualitative range 5 to 500 ng pL RNA 6000 Pico Kit Agilent Eukaryote Total RNA Pico Assay Qualitative range 50 to 5000 pg pL in water Small RNA Kit Agilent Small RNA Assay 50 to 200 pg miRNA 1to 10 ng pL of purified small RNA lt 200 nt 10 to 50 ng pL of total RNA miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 51 3 Characterization of Total RNA Step 2 Characterize the RNA samples using the Agilent 2100 Bioanalyzer To measure the total RNA integrity purity and concentration use one of the Agilent RNA 6000 kits Follow the instructions in the kit To measure the small RNA amount and profile use the Agilent Small RNA kit Follow the instructions in the kit 1 Ensure the 2100 bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 expert software version B 02 04 or higher switch on the 2100 bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the
13. A sample containing flow through on ice Check that the final flow through is translucent and slightly pink The flow through volume needs to be uniform across the samples and close to 50 uL Do not discard the final flow trough It now contains the labeled miRNA sample CAUTION 1 Step 3 Dry the sample After the 16 C labeling reaction or sample purification completely dry the samples Use a vacuum concentrator at 45 to 55 C or on the medium high heat setting This step may take up to 1 hour after column purification and up to 3 hours without column purification Check the samples after 30 minutes Continue vacuum concentration until they are dry To check for sample dryness flick hard on the tube and make sure that the pellets do not move or spread The sample must be completely dried after labeling Residual DMSO will adversely affect the hybridization results 24 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 4 Prepare the 10X Blocking Agent 1 Add 125 uL of nuclease free water to the vial containing lyophilized 10X GE Blocking Agent supplied with the Agilent miRNA Complete Labeling and Hyb Kit p n 5190 0456 2 Mix by gently vortexing 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 adhering to the tube walls or cap by centrifuging for 5 to 10 seconds at 17 000 x g 10X Blocking Agen
14. License for End Users Nucleotide Analog Component on page 60 for more licensing information Warranty The material contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent 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 merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance 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 doc ument that conflict with these terms the warranty terms in the separate 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 accor dance 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
15. Sept09 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 When the Agilent XDR scanned images are added to Feature Extraction software version 9 5 or later the High and Low images are automatically combined for data extraction 20 bit single images from the C Scanner are equivalent to 16 bit XDR images from the B Scanner 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 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 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol P
16. activity and thus the labeling efficiency CAUTION The Cyanine3 pCp is light sensitive Please minimize any exposure to light CAUTION 2 Immediately prior to use prepare the Ligation Master Mix by gently mixing the components listed in Table 7 and maintain on ice Table 7 Ligation Master Mix for T4 RNA Ligase Components Volume pL per reaction Volume pL per 9 reactions 10X T4 RNA Ligase Buffer 1 0 9 0 Cyanine3 pCp 3 0 27 0 T4 RNA Ligase 0 5 45 Total Volume 4 5 40 5 Be sure to use the Ligation Master Mix within 15 minutes of mixing all the components in Table 7 Failure to do so may affect the labeling efficiency 22 3 Immediately add 4 5 uL of the Ligation Master Mix to each sample tube for a total reaction volume of 11 3 uL 4 Gently mix by pipetting and gently spin down 5 Incubate at 16 C in a circulating water bath or cool block for 2 hours miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Procedures 2 Step 2 Purify the labeled RNA optional If you are not using the columns for purification continue at Step 3 Dry the sample on page 24 This step removes DMSO and free Cyanine3 pCp The use of the column reduces the needed drying time in Step 3 Dry the sample but it adds hands on processing time Micro Bio Spin 6 column preparation 1 Invert the column sharply several times to resuspend the settled gel and to remove any air bubb
17. agents 11 Required Hardware and Software 11 RNA Extraction Recommendation 13 2 Procedures Spike In Solution Preparation 17 To handle and store Spike In solutions 17 To prepare Spike In solutions 18 Sample Labeling and Hybridization 19 Step 1 Prepare the labeling reaction 19 Step 2 Purify the labeled RNA optional 23 Step 3 Dry the sample 24 Step 4 Prepare the 10X Blocking Agent 25 Step 5 Prepare hybridization samples 26 Step 6 Prepare the hybridization assembly 28 Microarray Wash 30 Step 1 Add Triton X 102 to Gene Expression wash buffers 30 Step 2 Prewarm Gene Expression Wash Buffer2 30 Step 3 Prepare the equipment 31 Step 4 Wash the microarray slides 32 Scanning and Feature Extraction 36 Step 1 Scan the slides 37 Step 2 Extract data using Agilent Feature Extraction Software 41 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Contents 3 Characterization of Total RNA Step 1 Prepare for characterization of total RNA 51 Step 2 Characterize the RNA samples using the Agilent 2100 Bioanalyzer 52 4 Reference Supplemental User Guides 56 Microarray Handling Tips 57 General Microarray Layout and Orientation 58 Array Sample tracking on an 8 pack array slide 59 Limited Use License for End Users Nucleotide Analog Component 60 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 1 Before You B
18. ake Figure 6 Feature Extraction version 9 5 dialog box 4 Check the Extraction Set Configuration For FE versions earlier than version 10 7 3 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 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 Template Browser and select Online Update You can also download the latest grid templates from Agilent Web site at www agilent com chem downloaddesignfiles After downloading you must add the grid templates to the Grid Template Browser miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 43 2 Procedures 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 c Verify that the correct protocol is assigned to each extraction set in the Protocol Name column For Agilent miRNA microarrays select miRNA_107_
19. at steps step 4 through step 6 for up to five additional slides in the group For uniform washing do up to a maximum of six disassembly procedures yielding six microarray slides 8 When all slides in the group are placed into the slide rack in slide staining dish 2 stir using setting 4 or a moderate speed setting for 5 minutes 9 Transfer slide rack to slide staining dish 3 containing Gene Expression Wash Buffer 2 at 37 C Stir using setting 4 or a moderate speed setting for 5 minutes You must maintain Wash 2 at 37 C for the duration of the wash step Failure to do so may result in alterations of stringency which may reduce the consistency of experimental results 10 Slowly remove the slide rack minimizing droplets on the slides It should take 5 to 10 seconds to remove the slide rack miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 33 2 Procedures 11 Discard the used Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 12 Repeat step 1 through step 11 for the next group of six slides using fresh Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 pre warmed to 37 C 13 Put the slides in a slide holder Figure 2 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 Agilent barcode facing up in a slide holder Make sure that the slide is not ca
20. canning Figure 11 Agilent microarray slide and slide holder 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 down In this orientation the active side containing the microarray is protected from potential damage by fingerprints and other elements Once securely placed the numeric barcode non active side of the slide is visible Figure 11 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 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Reference 4 Array Sample tracking on an 8 pack array slide Use the form below to make notes to track your samples on a 8 pack array slide Position the gasket slide in the SureHyb chamber base with the label to the left and load the samples top row left to right then lower row left to right The array suffix assignments from Feature Extraction will then occur in the order shown Arrays Array 1_1 Array
21. characterizes the total RNA sample with an emphasis on the small RNA content of which a fraction is miRNA This can be done using the Small RNA Kit along with the Small RNA assay on the Agilent 2100 bioanalyzer For assessment of total RNA quality the Agilent 2100 Expert Software automatically 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 For general assistance on the evaluation of total RNA with emphasis on the RNA integrity number refer to the application note RNA integrity number RIN Standardization of RNA quality control part number 5989 1165EN To download application notes and for more information regarding Agilent s 2100 bioanalyzer along with the various kits offered for analysis of both total RNA and small RNA visit the Web site at www agilent com chem labonachip 50 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Characterization of Total RNA 3 Step 1 Prepare for characterization of total RNA e Refer to Table 13 and Table 14 to make sure that you have the appropriate analyzer kits and compatible assays Table 13 Analyzer and Kits Description Company and part no 2100 Bioanalyzer RNA 6000 Nano Kit RNA 6000 Pico Kit Small RN
22. check the absorption spectrum from 220 nm to about 300 nm to detect some of these possible contaminants In the absorption spectra you should see only one peak with an absorption maximum at 260 nm You may also see a shoulder of an additional peak at lt 220 nm that overlaps with and inflates the absorption at 260 nm The level of inflation can be seen on the absorption spectrum If the absorption spectrum is not available calculate the ratio of absorbances at 260 230 nm This ratio should be greater than 1 8 If the ratio is less than 1 8 then you need to purify the sample further by doing additional extractions with chloroform followed by an ethanol precipitation To ensure recovery of the RNA do this purification with a large microgram quantity of total RNA Unfortunately absorption spectra cannot differentiate between RNA and DNA Contamination with DNA can result in overestimation of total RNA amounts and may lead to decreased sensitivity on the miRNA assay due to the effective decrease in the amount of total RNA added wit Agilent Technologies 49 3 Characterization of Total RNA Characterization of the input total RNA using the Agilent 2100 bioanalyzer provides information on the sample quality prior to labeling and hybridization You can use the RNA 6000 Nano kit to analyze total RNA with the appropriate assay at the assay specified concentration For low concentration samples use the RNA 6000 Pico kit The small RNA assay
23. directly into a sterile 1000 mL bottle Repeat until you have enough prewarmed Wash Buffer 2 solution for your experiment Tightly cap the 1000 mL bottle and place in a 37 C water bath the night before washing arrays Alternatively remove the plastic cubitainer from the box and place it in a 37 C water bath the night before washing the arrays Place slide staining dish 3 see Table 10 on page 32 for numbering of the wash dishes into a 1 5 L glass dish three fourths filled with water Warm to 37 C by storing overnight in an incubator set to 37 C miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 3 Prepare the equipment Always use designated clean equipment when doing the hybridization and wash steps Designate and dedicate dishes to miRNA experiments 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 steps 1 and 2 at least 5 times as it is necessary to remove any traces of contaminating material 4 Discard the Milli Q water CAUTION 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 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 31 2 32 Procedures Step
24. ed 3 53 3 Characterization of Total RNA Small RNA miRNA in total RNA The small RNA assay characterizes the total RNA sample with an emphasis on the small RNA content of which a fraction is miRNA The resulting electropherogram typically has a number of bands or peaks representing small RNAs ranging in size from 10 to 150 nt The miRNA portion is represented by the band or peak ranging in size from 10 to 40 nt These bands or peaks will vary in abundance depending on the total miRNA preparation Figure 10 shows an example of the resulting electropherogram of the small RNA assay for a total RNA sample You can record the results of the small RNA assay to correlate to your miRNA microarray data However the small RNA assay results may not necessarily be an indicator for the miRNA content in the sample and the miRNA microarray assay is a more sensitive technique for detecting low levels of miRNAs 5 10 20 30 40 60 100 150 nt Figure 10 Characterization of small RNA within a total RNA sample miRNA is typically found in the 10 to 40 nt region which is a subset of the small RNA that range from 10 to 150 nt 54 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol miRNA Microarray System with miRNA Complete Labeling and Hyb Kit 5 Protocol e 6 ee 4 e Reference Supplemental User Guides 56 Microarray Handling Tips 57 General Microarray Layout and Orientation 58 Array Sample tracking
25. egin Procedural Notes 8 Safety Notes 8 Agilent Oligo Microarray Kit Contents 8 Required Equipment 9 Required Reagents 10 Optional Equipment Reagents 11 Required Hardware and Software 11 RNA Extraction Recommendation 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 s microarrays App Agilent Technologies 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 use In general follow Biosafety Level 1 BL1 safety rules Safety Notes Dimethyl Sulfoxide DMSO may be harmful by inhalation ingestion or skin absorption DMSO vapor or mist is irritati
26. er Agilent GeneSpring software GX10 0 or later optional miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Before You Begin 1 RNA Extraction Recommendation Total RNA extraction methods differ in numerous ways and may impact e yield e the inclusion of small RNAs in the total RNA extraction e the quantification of the total RNA The Agilent miRNA Microarray system is for use with total RNA extracts Do not use size fractionation or small RNA enrichment protocols The following method has been validated on the Agilent miRNA Microarray system e Absolutely RNA miRNA Kit Agilent p n 400814 To get consistent results for comparative experiments use the same total RNA extraction method Different total RNA extraction methods can result in slightly different miRNA profiles Do not use purified miRNA This protocol supports the use of total RNA only Extraction methods that use organic solvents such as TRIZOL can result in inaccurate quantification because organic solvent contamination from carry over during the RNA extraction can compress the 260 230 ratio The affected 260 measurement can result in inaccurate quantification of the total RNA To ensure the removal of residual TRIZOL reagent do one to two additional chloroform extractions after the final extraction step in the TRIZOL protocol before you continue to the ethanol precipitation See Characterization of Total RNA on page 49 for m
27. first slide is located and the letter n represents the End slot where the last slide is located 40 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 2 Extract data using Agilent Feature Extraction Software Feature Extraction FE is the process by which information from probe features is extracted from microarray scan data allowing researchers to measure miRNA expression in their experiments To get the most recent Feature Extraction software for miRNA expression go to the Agilent Web site at www agilent com chem fe Refer to the Agilent Feature Extraction Software User Guide or Reference Guide for more information to use the software FE version 9 5 3 or higher is required for extraction of tif images of Agilent miRNA microarrays scanned on an Agilent Scanner FE version 10 7 gives you the option to automatically download new grid templates protocols and QC metrics QCM or QCMT via eArray and supports analysis of the spike ins miRNA See Automatic Download from eArray on page 47 for configuration Refer to Table 11 on page 36 to determine the version of Feature Extraction you need to use FE version 10 7 protocol uses surrogates in determining the gTotalGeneSignal which eliminates negative expression values Earlier versions of FE and FE protocols do not use surrogates and may create negative gTotalGeneSignal values 1 Open the Agilent Feature Extraction FE program To get the m
28. ilution solutions Add 2 uL from the 2nd Dilution Labeling Spike In tube into the 3rd Dilution Labeling Spike In tube Add 2 uL from the 2nd Dilution Hyb Spike In tube into the 3rd Dilution Hyb Spike In tube Mix well and briefly spin in a centrifuge This dilution is used in the labeling reaction or the hybridization reaction 7 Discard the 2nd Dilution and 3rd Dilution after use 8 Store 1st Dilution tubes at 80 C CAUTION Do not freeze thaw the original or 1st Dilution Spike In solution more than twice 18 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Sample Labeling and Hybridization Agilent s miRNA Complete Labeling and Hyb Kit p n 5190 0456 generates fluorescently labeled miRNA with a sample input of 100 ng of total RNA This method involves the ligation of one Cyanine 3 pCp molecule to the 3 end of a RNA molecule with greater than 90 efficiency The Agilent miRNA Complete Labeling and Hyb Kit provides all the needed components for sample labeling and hybridization preparation These include Calf Intestinal Alkaline Phosphatase CIP 10X Calf Intestinal Phosphatase Buffer T4 RNA Ligase 10X T4 RNA Ligase Buffer Dimethyl Sulfoxide DMSO Nuclease Free Water Cyanine 3 pCp 10X GE Blocking Agent 2X Hi RPM Hybridization Buffer Please refer to Characterization of Total RNA on page 49 for procedural recommendations on total RNA characterization Read the pro
29. les Snap off the tip and place into a 2 mL microcentrifuge tube supplied with the columns from Bio Rad Remove the green cap from the column If the buffer does not drip into the 2 mL microcentrifuge tube press the green cap back onto the column and remove it again Let the buffer drain for about 2 minutes 4 Check to make sure that all columns are evenly drained Discard the drained buffer from the 2 mL microcentrifuge tube and then place the column back into the tube Spin the microcentrifuge tube containing the column for 2 minutes at 1000 x g in a centrifuge The speed of the microcentrifuge must be accurately set for example 1000 x g not 1000 rpm If the spin speed is too low sample can be lost If the spin speed is too high desalting may be ineffective and the column may break down 7 Remove the column from the 2 mL microcentrifuge tube and place it into a clean 1 5 mL microcentrifuge tube Discard the 2 mL microcentrifuge tube miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 23 2 Procedures CAUTION Sample purification 1 Add 38 7 uL of RNase Free Water or 1X TE pH 7 5 to the labeled sample for a total volume of 50 uL Without disturbing the gel bed pipette the 50 uL sample onto the gel bed from step 7 above Elute the purified sample by spinning the microcentrifuge tubes containing the columns for 4 minutes at 1000 x g in a centrifuge Discard the columns and keep the miRN
30. lete Labeling and Hyb Kit Agilent p n 5190 0456 Gene Expression Wash Buffer Kit Agilent p n 5188 5327 Milli O water or equivalent miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Before You Begin 1 Optional Equipment Reagents Table 3 Description Company and part no MicroRNA Spike In Kit Agilent p n 5190 1934 Universal miRNA Reference Kit Agilent p n 750700 2100 Bioanalyzer Agilent p n G2938A RNA 6000 Nano Kit Agilent p n 5067 1511 RNA 6000 Pico Kit Agilent p n 5067 1513 Ozone Barrier Slide Covers Agilent p n G2505 60550 Small RNA Kit Agilent p n 5067 1548 MicroBioSpin 6 Columns Bio Rad p n 732 6221 Slide box Corning p n 07201629 PCR 96 plate Eppendorf p n 951020401 Dye filter Sigma p n 2361569 Thermocycler Required Hardware and Software Table 4 Description Agilent G4450AA Feature Extraction software 9 5 or later without Spike In or Feature Extraction 10 7 3 with Spike In Agilent Scan Control software version A 7 0 or later includes XDR functionality Pentium IV 1 5 GHz or higher 512 MB RAM 1 GB recommended 20 GB available disk space if saving images and results files locally miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 11 1 12 Before You Begin Table 4 Description Microsoft Windows 2000 with SP2 or later fully tested on SP4 Windows XP SP2 Microsoft Internet Explorer 5 5 or later Adobe Acrobat Reader 4 0 or lat
31. mat and AgilentG3_miRNA for the 8x60K format All the appropriate settings are set as default and you need not change anything else See Figure 4 for the version 8 x Scan control dialog box Make sure you scan 8x60K microarrays at 3 microns Feature Extraction cannot extract CAUTION f the data if G3 microarrays are scanned at 5 microns If neither profiles are on your local system download them from www agilent com chem scanner under Download Software Download and unzip the profile Within the Scan Control Software select Tools gt Profile Editor gt Import then browse for the location of the scan profile file and select Open Two profiles will then be added to the available list of profiles miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 37 2 38 Procedures Step 1 Scan the slides EX Agilent Scan Control File Tools Help Start slot 1 X Endslot 48 v Profile A X Slide ID Channels Scan Region Resolution TIFF cb Agilent HD 61 x 21 6mm Sum 20 bit lt Auto detect gt Agilent HD 61 x 21 6mm Sum 20 bit lt Auto detect gt Agilent HD 61 x 21 6mm Sum 20 bit lt Auto detect gt Agilent HD 61 x 21 6mm Sum 20 bit lt Auto detect gt Agilent HD 61 x 21 6mm Sum 20 bit lt Auto detect gt Agilent HD 61 x 21 6mm Sum 20 bit RA a ress OFF Split Rotate Off Naming lt Instrument Serial gt _ lt Slic Total file size 7 1 GB Total run time 6 Figure
32. miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol For use with Agilent miRNA microarrays Version 2 4 September 2011 Microarrays manufactured with Agilent SurePrint Technology Research Use Only Not for use in Diagnostic Procedures Ee Agilent Technologies Notices Agilent Technologies Inc 2010 2011 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 64170 90011 Edition Version 2 4 September 2011 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgements Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated Pentium is a U S registered trademark of Intel Corporation Windows is a registered trademark of Microsoft Corporation in the U S Technical Support For technical product support contact your local Agilent Support Services representa tive You can get Agilent s worldwide sales and support center telephone numbers at www agilent com chem contactus or send an email to genomics agilent com Limited Use License for End Users For Internal Research Use only Not for use in diagnostic procedures See Limited Use
33. nd record the time Record whether bubbles formed during hybridization and if all bubbles are rotating freely Make note of any significant loss of hybridization volume miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Procedures 2 5 Prepare the hybridization chamber disassembly a Place 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 Remove the microarray slide and place into the slide rack in 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 7 Repe
34. ng to the eyes mucous membranes and upper respiratory tract It can cause skin irritation and allergic respiratory and skin reactions Overexposure may cause reproductive disorder s based on tests with laboratory animals 2X Hi RPM Hybridization Buffer is considered hazardous by the OSHA Hazard Communication Standard 29 CFR 1910 1200 It may be harmful if swallowed The buffer contains material that may cause damage to the skin and central nervous system Please refer to the MSDS for complete safety information Agilent Oligo Microarray Kit Contents e Eight microarrays printed on a 1 inch x 3 inch glass slide one or three slides per kit e CD containing microarray design files in various file formats miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Before You Begin 1 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 for any species in the miRBASE 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 N purge box Do not store microarray slides in open air after breaking foil Required Equipment Table 1 Required equipment Description Company and part no Microarray Scanner Hybridization Chamber stainles
35. nning and Feature Extraction 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 on an 8 pack array slide on page 59 for guidelines on tracking sample position for multipack slide formats If you are not processing all of the arrays on a slide place unused gasket wells at the far end opposite of the barcode In the unused wells pipette 45 uL of 1X hybridization buffer 2 Slowly dispense all of the volume of the hybridization sample onto the gasket well in the center Heating and cooling the hybridization sample results in varying degrees of volume loss To adjust for the variation in final sample volume adjust your pipette to avoid the introduction of air bubbles during application to the gasket well To avoid leakage from the gasket well minimize the introduction of bubbles during sample loading Do not allow the pipette tip or the hybridization solution to touch the gasket walls Allowing liquid to touch the gasket wall greatly increases the likelihood of gasket leakage 28 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Procedures 2 3 Slowly place an array active side down onto the SureHyb gasket slide so that the Agilent labeled barcode is facing down and the numeric barcode is facing up Verify that
36. on an 8 pack array slide 59 Limited Use License for End Users Nucleotide Analog Component 60 This chapter contains reference information related to the protocol and the limited use license ois Agilent Technologies 55 4 56 Reference Supplemental User Guides First time users of Agilent s oligo microarray system please refer to the following user guides for detailed descriptions and operation recommendations for each of the hardware and software components used in the miRNA platform workflow The user guides were included with your Agilent hardware and software You can also download them from the Agilent web site at www agilent com chem dnamanuals protocols Search on publication type Manuals e G2534A Microarray Hybridization Chamber User Guide e G2545A Hybridization Oven User Manual e G2565CA or G2565BA Microarray Scanner System User Guide or the G4900DA SureScan Microarray Scanner User Guide e Feature Extraction Installation Guide e Feature Extraction Quick Start Guide e Feature Extraction User Guide e Feature Extraction Reference Guide miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol CAUTION Reference 4 Microarray Handling Tips Each microarray is printed on the side of the glass slide containing the Agilent labeled barcode This side is called the active side The numeric barcode is on the inactive side of the slide You must familiarize yourself with the a
37. ore information on RNA purity and quality assessment miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 13 1 Before You Begin RNA Extraction Recommendation 14 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol miRNA Microarray System with miRNA Complete Labeling and Hyb Kit 5 Protocol e 6 ee 2 e Procedures Spike In Solution Preparation 17 To handle and store Spike In solutions 17 To prepare Spike In solutions 18 Sample Labeling and Hybridization 19 Step 1 Prepare the labeling reaction 19 Step 2 Purify the labeled RNA optional 23 Step 3 Dry the sample 24 Step 4 Prepare the 10X Blocking Agent 25 Step 5 Prepare hybridization samples 26 Step 6 Prepare the hybridization assembly 28 Microarray Wash 30 Step 1 Add Triton X 102 to Gene Expression wash buffers 30 Step 2 Prewarm Gene Expression Wash Buffer 2 30 Step 3 Prepare the equipment 31 Step 4 Wash the microarray slides 32 Scanning and Feature Extraction 36 Step 1 Scan the slides 37 Step 2 Extract data using Agilent Feature Extraction Software 41 Agilent s miRNA Microarray System uses cyanine 3 labeled targets to measure miRNA in experimental and control samples Figure 1 is a standard workflow for sample preparation and array hybridization wit Agilent Technologies 15 2 Procedures Total RNA 100 ng Labeling Spike In optional y Phosphatase Treatment incubate 30 minutes 37 C Dephosphoryla
38. ost recent Feature Extraction protocols for miRNA go to the Agilent Web site at 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 an Agilent scanner For auto assignment of the miRNA FE protocol the default miRNA 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 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 41 2 42 Procedures 3 Set FE Project Properties Select the Project Properties tab In the General section enter your name in the Operator text box In the Output section verify that at least the following default settings as shown in Figure 6 on page 43 are selected For versions of FE earlier than 10 7 3 in the Other section from the QC Metric Set drop down list select miRNA_QCMT_Jan09 as shown in Figure 6 on page 43 If the QC Metric Set is not a
39. p miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 7 Incubate at 100 C for 5 minutes 8 Immediately transfer to an ice water bath for 5 minutes CAUTION Do not leave samples in the ice water bath for more than 15 minutes Longer incubations may adversely affect the hybridization results 9 Quickly spin in a centrifuge to collect any condensation at the bottom of the tube 10 Immediately proceed to Step 6 Prepare the hybridization assembly on page 28 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 27 2 Procedures CAUTION Step 6 Prepare the hybridization assembly Refer to the Agilent Microarray Hybridization Chamber User Guide G2534 90002 for in depth instructions on how to load slides assembly and disassembly of chambers as well as other helpful tips This user guide is available with the Agilent Microarray Hybridization Chamber Kit G2534A and can also be downloaded from the Agilent Web site at www agilent com chem dnamanuals protocols Go to http www genomics agilent com and search on Running a Microarray Experiment to view an instructional video To process the miRNA system microarray 1 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 Ensure that the gasket slide is flush with the chamber base and is not ajar For Agilent sca
40. rocedures 2 If a QC Metric Set has been assigned to the FE Project you can view the results of the metric evaluation in three ways e Project Run Summary includes a summary sentence QC Report includes both a summary on the header and a table of metric values Figure 7 shows a QC report for an miRNA microarray with the use of spike in miRNAs QC Chart includes a view of the values of each metric compared across all extractions in FE Project Page 2 of 2 QC Report Agilent Technologies miRNA Date Thursday November 04 2010 12 34 Grid Foreground Surface Fit Spatial Distribution of Median Signals for each Row 030840_D_F 20101103 Image us23502418_253084010001_S01 1_1 BG Method No Background RMS_Fit RMS_Resid Avg_Fit Protocol miRNA_107_Sep09 Read Only Background Detrend On FeatNCRange LoPass User Name pdandrad Multiplicative Detrend False Reproducibility CV for Replicated Probes Median CV Signal inliers Non Control probes FE Version 10 7 3 1 Additive Error 3 Sample red green Saturation Value 777710 Spot Fi f the Four Corners of the Array BGSubSignal 4 96 ProcessedSignal 4 96 Net Signal Statistics Non Control probes wae Local ne Background Non Uniform 2 Population 2103 Spatial Distribution of All Outliers on the Array 4 rows x 164 columns FeatureNonUnif 2 0 00 Feature Population Outliers 2103 4 11 Figure 7 Saturated Features 99 of
41. s Hybridization Chamber gasket slides 8 microarrays slide 5 slides box Hybridization oven 20 rpm capability temperature set at 55 C Hybridization oven rotator for Agilent Microarray Hybridization Chambers Nuclease free 1 5 mL microcentrifuge tubes Magnetic stir bar x2 Magnetic stir plate Magnetic stir plate with heating element Microcentrifuge Slide staining dish with slide rack x3 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 Eppendorf p n 022363204 Corning p n 401435 or equivalent Corning p n 6795 410 or equivalent Eppendorf p n 5417R or equivalent Thermo Shandon p n 121 or equivalent miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 9 1 10 Before You Begin Table 1 Required equipment continued Description continued Company and part no Circulating water baths or heat blocks set to 16 C 37 C and Corning p n 6795 420 or 100 C equivalent Vacuum concentrator with heater Savant SpeedVac p n SPD111V or equivalent 1 5L glass dish Pyrex p n 7211 or equivalent Clean forceps Ice bucket Pipetman micropipettors P 10 P 20 P 200 P 1000 or equivalent Powder free gloves Sterile nuclease free aerosol barrier pipette tips Vortex mixer Timer N3 purge box for slide storage Required Reagents Table 2 Description Company and part no miRNA Comp
42. ssembly and disassembly instructions for use with the Agilent Microarray Hybridization Chamber G2534A and gasket slides 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 placed 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 Do not write anywhere on the array or gasket slide as you can introduce severe artifacts on the scanned images miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 57 4 58 General Microarray Layout and Orientation Agilent oligo microarray 8 microarray slide format as imaged on Agilent microarray scanners Microarrays are printed on the side of the glass labeled with the Agilent bar code also called the active side or front side ILAA AA Ala Agilent microarray slide holder for SureScan above and Scanner B C below microarray scanners i s2 gt gt a mE gm H Agilent Microarray Scanner scans through the glass Back side s
43. t can be prepared in advance and stored at 20 C for up to 2 months After thawing repeat the vortexing and centrifugation procedures before use miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 25 2 26 Step 5 Prepare hybridization samples Equilibrate water bath or heat block to 100 C 2 Resuspend the dried sample in 17 uL of nuclease free water when the Spike In solution is used and 18 uL when the Spike In solution is not used 3 Optional Add 1 0 uL of the Hyb Spike In solution 8rd Dilution to each sample 4 Add 4 5 uL of the 10X GE Blocking Agent to each sample 5 Add 22 5 uL of 2X Hi RPM Hybridization Buffer to each sample for a total of 45 uL 6 Mix well but gently on a vortex mixer Table 8 Hybridization mix for miRNA microarrays with Hyb Spike In solution Components Volume pL Labeled miRNA sample 17 Hyb Spike In 1 10X GE Blocking Agent 45 2X Hi RPM Hybridization Buffer 22 5 Total Volume 45 Table9 Hybridization mix for miRNA microarrays without Hyb Spike In solution Components Volume pL Labeled miRNA sample 18 10X GE Blocking Agent 45 2X Hi RPM Hybridization Buffer 22 5 Total Volume 45 For best results stagger the tubes in sets of eight such that eight arrays on one array slide can be loaded at once Have the SureHyb chamber along with the gaskets and microarrays easily available to prepare the hybridization assembly before you continue to the next ste
44. ted RNA V Add DMSO y Heat ice Assemble Labeling Reaction incubate 2 hours 16 C Labeled RNA y _ Desalt with Spin Column optional Desalted Labeled RNA Dry sample with vacuum concentrator approximately 2 to 3 hours 1 hour with columns 45 C to 55 C A y Assemble Hybridization Mixture Hyb Spike In optional y Heat ice v Hybridize 20 hours 55 C 20 RPM v Wash Scan miRNA Profile The sample can be stored at 80 C after this step if needed Figure 1 Workflow for sample preparation and array processing with the optional Bio Rad columns and spike ins 16 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Spike In Solution Preparation Agilent s microRNA Spike In kit p n 5190 1934 consists of two microRNA Spike In solutions for process control Use the Spike In solutions to help distinguish significant biological data from processing issues The Labeling Spike In solution is to be spiked into the labeling reaction while the Hyb Spike In solution is to be spiked into the hybridization reaction To handle and store Spike In solutions CAUTION Do not use spike in solutions for normalization purposes Do not use with drosophila miRNA microarrays Spike in solutions are derived from drosophila sequences 1 Thaw all components 2 Briefly spin tubes 3 Label 5 tubes 1st Dilution Labeling Spike In and an additional 5 tubes 1st Dilution Hyb Spike In
45. the sandwich pair is properly aligned When you assemble the array slide to the gasket slide keep the array slide parallel to the gasket slide at all times and do not drop the array slide onto the gasket slide Doing so increases the chances of samples mixing between gasket wells CAUTION 4 Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces 5 Firmly hand tighten the clamp onto the chamber 6 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 7 Place assembled slide chamber in rotisserie in a hybridization oven set to 55 C Set your hybridization rotator to rotate at 20 rpm 8 Hybridize at 55 C for 20 hours Be sure that the arrays are hybridized for at least 20 hours Hybridization can occur for longer than 20 hours but the actual hybridization time should be consistent if the results are to be compared Failure to maintain consistent hybridization times may adversely affect your data 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 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 b
46. tocol in its entirety before you continue Step 1 Prepare the labeling reaction This protocol is compatible with hybridization of labeled miRNA to either 8x15K or 8x60K microarray formats Dephosphorylation 1 Dilute total RNA sample to 50 ng uL in 1X TE pH 7 5 or DNase RNase free water 2 Add 2 uL 100 ng of the diluted total RNA to a 1 5 mL microcentrifuge tube and maintain on ice miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 19 2 20 Procedures 3 Immediately prior to use prepare the Calf Intestinal Alkaline Phosphatase CIP Master Mix from Table 5 when using the Labeling Spike In solution 3rd Dilution Table 6 when not using the Labeling Spike In solution Add the components in the order indicated and maintain on ice Table5 CIP Master Mix with Labeling Spike In solution Components Volume pL per reaction Volume pL per 9 reactions 10X Calf Intestinal 0 4 3 6 Phosphatase Buffer Labeling Spike In 1 1 9 9 Calf Intestinal Phosphatase 0 5 45 Total Volume 2 0 18 0 Table 6 CIP Master Mix without Labeling Spike In solution Components Volume pL per reaction Volume pL per 9 reactions 10X Calf Intestinal 0 4 3 6 Phosphatase Buffer Nuclease Free Water 1 1 9 9 Calf Intestinal Phosphatase 0 5 4 5 Total Volume 2 0 18 0 4 Add 2 uL of the CIP Master Mix to each sample tube for a total reaction volume of 4 uL Gently mix by pipetting 5 Dephosphorylate the sample
47. tocol_Array_Num txt for example US22502705_251911810634_S01_miRNA v1_95_May07_1_1 txt includes all feature probe and gene level data as well as array level statistics and parameters Use these files for analysis and to review the QC metrics in the GeneSpring GX version 10 or later The other text file the GeneView file uses the name BarcodeNum_SO _Array_Num_GeneView txt for example 251911810634_S01_1_1_GeneView txt The GeneView file provides the summarized TotalGeneSignal TotalGeneError and a detection flag for each miRNA gene This simple file is appropriate for any analyses and can be loaded into GeneSpring GX version 9 0 or similar software for analysis However it does not contain the QC metric data for review The gTotalGeneSignal column has been background subtracted and outlier rejected and is appropriate for use as the measured intensity for each miRNA gene without further manipulation Please refer to the Agilent Feature Extraction Software User Guide or Reference Guide for more detail on the data summarization algorithms and for information on other columns in the feature extraction result files miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 2 Extract data using Agilent Feature Extraction Software Automatic Download from eArray Feature Extraction version 10 7 or later can automatically download Grid Templates protocols and QC metrics QCM or QCMT
48. traction GeneSpring Format version Control version version version 8x60K SureScan 9 X AgilentG3_miRNA Yes 10 7 3 or later 11 5 or later 8x60K SureScan 9 X AgilentG3_miRNA No 10 5 or later 10 0 or later 8x15K SureScan 9 X AgilentHD_miRNA Yes 10 7 3 or later 11 5 or later 8x15K SureScan 9 X AgilentHD_miRNA No 10 5 or later 10 0 or later 8x60K c 8 X AgilentG3_miRNA Yes 10 7 3 or later 11 0 or later 8x60K C 8 X AgilentG3_miRNA No 10 5 or later 10 0 or later 8x15K C 8 X AgilentHD_miRNA Yes 10 7 3 or later 11 0 or later 8x15K C 8 X AgilentHD_miRNA No 10 5 or later 10 0 or later 8x15K B 7X See Table 12 on Yes 10 7 3 or later 11 0 or later page 39 8x15K B 7X See Table 12 on No 9 5 3 or later 10 0 or later page 39 36 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Step 1 Scan the slides Agilent Scanner Settings Make sure the scanner has been turned on for at least 15 minutes before you continue 1 Assemble the slides into a SureScan or Scanner C or B slide holder Place the slides into the slide holder such that the numeric barcode side is visible not the Agilent labeled barcode side Refer to General Microarray Layout and Orientation on page 58 2 Place assembled slide holders into either the SureScan cassette or Scanner B or C carousel 3 Verify scan settings for miRNA If you use a SureScan or Scanner C scanner then select the protocol or profile AgilentHD_miRNA for the 8x15K for
49. uffer the night before your planned microarray wash step Do this in two steps Step 1 Add Triton X 102 to Gene Expression wash buffers on page 30 and Step 2 Prewarm Gene Expression Wash Buffer 2 on page 30 the night before you wash the microarray slides miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 29 2 Procedures Microarray Wash 30 Step 1 Add Triton X 102 to Gene Expression wash buffers Add the Triton X 102 to Gene Expression wash buffer 1 and 2 when the cubitainer of wash buffer is first opened Do this step to both Gene Expression wash buffer 1 and 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 Use a pipette to add 2 mL of the provided 10 Triton X 102 into the wash buffer in the cubitainer Replace the original inner and outer caps and mix the buffer carefully but thoroughly by inverting the container 5 to 6 times Carefully remove the outer and inner caps and install the spigot provided with the wash buffer Prominently label the wash buffer box to indicate that Triton X 102 has been added and indicate the date of addition Step 2 Prewarm Gene Expression Wash Buffer 2 The temperature of the Gene Expression Wash Buffer must be at 37 C for optimal performance Warm the Gene Expression Wash Buffer 2 to 37 C as follows 1 Dispense 1000 mL of Gene Expression Wash Buffer 2
50. ught up on any corner Put an ozone barrier slide cover on top of the array as shown in Figure 3 Figure 3 Inserting the ozone barrier slide cover for Scanner B and C In environments in which the ozone level is below 50 ppb put the slides with Agilent barcode facing up in a slide holder 14 Scan slides immediately to minimize the impact of environmental oxidants on signal intensities If necessary store slides in orange slide boxes in a N purge box in the dark Use fresh Gene Expression Wash Buffer 1 and 2 for each wash group up to 6 slides 34 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 The wash buffer in the disassembly dish may be pink due to the presence of unincorporated cyanine 3 dye Discard the used wash buffer according to appropriate institutional guidelines You can use activated charcoal to absorb the unincorporated cyanine 3 dye from the used wash buffer if needed Extractors from Sigma p n 2361569 can be used for this purpose miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 35 2 Procedures Scanning and Feature Extraction This section describes how to scan and extract data from miRNA microarrays Refer to the Agilent Microarray Scanner User Guide for more information on how to use the scanner Table 11 Scanner to GeneSpring Workflow version compatibility Microarray Scanner Scan Scan settings profile Spike In Feature Ex
51. vailable to select from the pull down menu you must add it to the QC Metric Set Browser To add right click inside the QC Metric Set Browser and click Add Browse for the QC Metric Set file and click Open to load the QC Metric Set into the FE database If you don t have a local copy of the QC Metric Set or would like to download the latest QC Metric Set you can do so from Agilent Web site at www agilent com chem feqcemetrics After downloading you must add the QC Metric Set to the QC Metric Set Browser After a new QC Metric Set is added to the QC Metric Set Browser remember to specify the default protocol for the new QC Metric Set if you want the Feature Extraction program to automatically assign a FE QC Metric Set to an extraction set miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol Procedures 2 Qs Project Properties lo Extraction Sat Configuration B General Operator Unknown B Input Number of Extraction Sets Included 1 B Output and Data Transfer B Outputs E MAGE Local file only FEG Visual Rests Dea fhe erty Gid Local file only QC Report Local file only FTP Send Tiff Fle False E Local File Folder Sane As Image False Raste Folder iteng E FTP Setting E Automatic Protocol Assignment Highest Priority Defaut Protocol Grid Template Defaut Project Defauk Protocol E Automatic Grid Template Assignment Use Grid file if available Fake X Extern Oeo Overwrite Previous Results F
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