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TaqMan® Array Micro Fluidic Cards

1. 26 TaqMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card 2 Array Information CD Array Map File Contents An Array Map File contains two color coded maps that show the position of the assays on the TagMan Array card The top map shows the replicate distribution of assay gene symbols for each well The bottom map shows the TaqMan Gene Expression Assay ID numbers Array Map Files also indicate the TagMan Array card configuration and part number Production number of the TaqMan Array card Each TagMan Array card is assigned a unique production number This number appears as part the AIF Array Map File and SDS Setup File names Formats Each Array Information CD contains two Array Map Files one in HTML format and the other in spreadsheet format To open using View a map prodNum cardmap html your web browser Print a map prodNum cardmap xls Microsoft Excel software Where prodNum is the production number of the TaqMan Array card Assay Information File AIF About the AIF The Assay Information File AIF is a text file that describes the TagMan Array card AIF ProdNum txt where ProdNum is the manufacturing production number To view the AIF as a spreadsheet in Microsoft Excel Software 1 Load the Array Information CD into the CD drive 2 Navigate to the drive tha
2. Title Part Number Amplification Efficiency of TaqMan Gene Expression Assays 127AP05 Applied Biosystems 7900HT Fast Real Time PCR System and SDS 4351684 Enterprise Database User Guide Applied Biosystems 7900HT Fast Real Time PCR System Relative 4364016 Quantitation Using Comparative C Getting Started Guide Applied Biosystems 7900HT Fast Real Time PCR System Site Preparation 4351923 Guide Applied Biosystems Real Time PCR Systems Reagent Guide 4387787 High Capacity cDNA Reverse Transcription Kits Protocol 4375575 High Capacity RNA to cDNA Kit Protocol 4387951 High Capacity RNA to cDNA Master Mix Protocol 4377474 TagMan Gene Expression Assays Protocol 4333458 TaqMan Gene Expression Cells to C Kit Protocol 4385117 TagMan Gene Expression Master Mix Protocol 4371135 TaqMan PreAmp Master Mix Kit Protocol 4384557 TaqMan Universal PCR Master Mix Protocol 4304449 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Using TaqMan Endogenous Control Assays to Select an Endogenous 127AP08 Control for Experimental Studies Application Note vi TagMan Array Micro Fluidic Cards User Guide Obtaining Information from the Help System Send Us Your Comments Preface How to Obtain Support The SDS software Help system describes how to use each feature of the user interface To access the Help system you can Click in the toolbar Select Help SDS Online Help Press F1 To find top
3. SNP Type Not applicable to TagMan Gene Expression Assay N A Minor Allele Frequency Caucasian Minor Allele Frequency African American Minor Allele Frequency Japanese Minor Allele Frequency Chinese Celera Assembly Build Number Location on Celera Assembly NCBI Assembly Build Number Location on NCBI Assembly Notes 30 TaqMan Array Micro Fluidic Cards User Guide Chapter 3 Prepare Your Samples This chapter covers B Prepare Your Samples kk kK KK KK KK KK KK KK KK n 32 B Store the cDNA Samples uk KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK 34 TaqMan Array Micro Fluidic Cards User Guide 31 Chapter 3 Prepare Your Samples Prepare Your Samples Prepare Your Samples Isolate total RNA from your samples this page v Evaluate the RNA page 33 gt Perform reverse transcription RNA to cDNA page 34 RNA Template Guidelines Isolate Total RNA Notes Note When preparing your samples see the product part numbers and sizes under For Preparing Your Samples on page 60 For optimal performance Applied Biosystems recommends using RNA with the following characteristics Between 0 002 and 0 2 ug uL in concentration of RNA Less than 0 005 of genomic DNA by weight Free of inhibitors of reverse transcription and PCR Dissolved in PCR compatible buffer Free of RNase activity Note If y
4. TaqMan Array Micro Fluidic Cards User Guide 5 4 Chapter 1 Introduction User Guide Overview User Guide Overview Notes Workflow 10 minutes Prepare the card Prepare your samples Run the card Analyze the experiment YA Isolate the total RNA AVE Sa Az A Evaluate the RNA SHS BD v 5 s mRNA Perform reverse transcription 3 cic Prepare the PCR reaction mix Add the reaction mix to the card Centrifuge the card Seal the card Set up the experiment SDS Software w x5 RT primer s __ 5 cDNA Perform the run Configure the analysis settings RQ Study Manager or AAC Study Review the results Optional Perform downstream analysis NN For example TaqMan Gene Expression Cells to C M Kit High Capacity RNA to cDNA Kit or High Capacity cDNA Reverse Transcription Kit A TagMan master mix TaqMan Array Micro Fluidic Card System software and information CD 7900HT Fast Real Time PCR System with a TaqMan Array Micro Fluidic Thermal Cycling Block System software M TagMan Array Micro Fluidic Cards User Guide For More Chapter 1 Introduction 1 User Guide Overview Information Products For product part numbers and sizes see Appendix C on page 59 Documentation For document titles and part numbers see How to Obtain More Information on page vi
5. A f GR2163 After Run Analysis Amplification Plots are noisy across portions of the TagMan Array card In the Multicomponent Plot the FAM and ROX dye signals are below the background for the affected wells No amplification across large portions of the TaqMan Array card In the Multicomponent Plot the FAM and ROX dye signals are below the background The TaqMan Array card is misaligned Check the TagMan Array card for in the TagMan Array Micro Fluidic crushed or distorted feet If there are Card Thermal Cycling Block crushed feet contact Applied Biosystems Technical Support or contact your Applied Biosystems Field Applications Specialist FAS No amplification The Multicomponent Plot shows a rapid drop in ROX dye signal Empty wells due to improper staking Use a steady even motion when using the staker Notes 54 TagMan Array Micro Fluidic Cards User Guide Appendix A Troubleshooting Observation Possible Cause Recommended Action No amplification within or across one or more rows within a single fill reservoir The Multicomponent Plot shows a rapid drop in ROX dye signal Empty wells due to improper staking Use a steady even motion when using the staker Empty wells due to misalignment of the staker stylus If the staker stylus is misaligned contact your Applied Biosystems FAS No master mix loaded in the fill reserv
6. Services and Support For the latest services and support information see How to Obtain Support on page vii Notes TaqMan Array Micro Fluidic Cards User Guide 1 Chapter 1 Introduction User Guide Overview Notes 8 TaqMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card This chapter covers E Order the TagMan Array Micro Fluidic Card KK KK KK 10 B Available Formats KK KK KK KK KK KK KK KK kk kk kk kk 22 B Array Information CD Lc ku k ah ekle l d ka eee a lk all daha 26 TaqMan Array Micro Fluidic Cards User Guide 9 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card Order TaqMan Array Custom Order TagMan Gene Signature gt Reorder TaqMan Array Custom Micro Micro Fluidic Cards or TagMan9 Arrays page 16 Fluidic Cards or TagMan Gene Sets Gene Sets this page page 19 Order Taq Man9 1 Goto www appliedbiosystems com Array Custom Micro Fluidic 2 In the I Want to Buy box click TaqMan Gene Expression Assays Plates amp Cards or Arrays TaqMan Gene Sets O TaqgMan Gene Expression Assays Plates amp Arrays TaqMan MicroRNA Assays TaqMan SNP Genotyping Assays Real Time PCR Reagents amp Kits Custom Primers amp Probes As
7. 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card Ordering Information Product Description Literature Support The TagMan Human ABC Transporter Array contains assays for 50 human genes in addition to 14 endogenous controls in a 384 NE well array You can view your contract pricing for products in your Shopping Basket or Favorites My Basket11 2 v My Favor v Add to Favorites List Price Product Name Part Number Bang View Package Your Price 9a TagMan 4378700 4 cards Human ABC Transporter Array 9b My Basket11 2 Y JIBER DE LOSS My Favor y Add to Favorites 9c Note See user s manual or package insert for limited label license and trademark information For Research Use Only Not for use in diagnostics procedures Trademark c Click Add to Basket You can Repeat step 3 on page 16 through step 9 on page 17 to continue ordering TaqMan Gene Signature Arrays Click Shopping Basket to check out EJ Your order was successfully added to your Shopping Basket Applied Biosystems ships the TaqMan Array cards at ambient temperature Upon receipt store the cards at 2 to 8 C The cards are stable for up to 2 years from the manufacturing date Notes 18 TaqMan Array Micro Fluidic Cards User Guide Reorder TaqMan Array Custom Micro Fluidic Cards or TaqMan Gene Sets Cha
8. Applied Bibbystems Part Number 4400263 Rev B
9. configure a product or to view products available for purchase in your country O Product Name Part Number Quantity Package TaqMan amp Array LDA 1 cards L Configure 6 On the Store Log In page enter your user name and password Note If you are already logged in the Store Log In page does not appear Go to the next step 7 In step 1 of the Configurator click Import Saved Design Ordering Information Product Description Literature Support Related Products Configure Configuration gy Import Saved pg Compatible Tools Design Instruments Select a Format Select a Format for your TaqMan Array and then click Next to proceed To see a visual representation of a particular array format click View Array Layout To see a layout for all array formats in one document please download the All_Array_Maps pdf 57Kb cribe Your TaqMan Array Name E Description Required Notes 20 TaqMan Array Micro Fluidic Cards User Guide 8 9 Chapter 2 Order the TagMan Array Micro Fluidic Card 2 Order the TaqMan Array Micro Fluidic Card On the Designs page click the name of the design you want to import You are returned to step 4 of the Configurator Review amp Order Designs oo REZEN Design files for Tagman Arrays and SNPlex are stored and summarized below Your total workspace storage limit for both Designs and Saved Lists is 10000KB Design files
10. The same threshold setting must be used for an assay across all samples or TaqMan Array cards within a study Note You can use either TaqMan Universal PCR Master Mix or TaqMan Gene Expression Master Mix in your experiment However Applied Biosystems strongly recommends that you use only one type of master mix per study 2 In the well table or results table review the C4 values for each well and for each replicate group If needed omit outliers 50 TaqMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Analyze the Experiment 3 Review the gene expression plot for SDS Software v2 3 view the amplification plots in the Plate Detector or Sample view Note Relative gene expression measurements within and across manufacturing lots of TaqMan Array cards are reproducible You can compare normalized data AC values but not raw C values IMPORTANT If you are using different lots of TaqMan Array cards with the same set of assays be sure to use the correct SDS Setup File Different lots with the same set of assays may ship with different SDS Setup Files Optional For further data analysis the raw C and the AC values can be exported If you are using Perform SDS Software v2 3 you can export the values from the Plate Centric view Downstream De j Analvsis For detailed downstream analysis Applied Biosystems recommends the Real Time y StatMiner Software For information refer t
11. pl Name amp Describe Your TaqMan Array Name I Description Import Saved Design Required Minimum amp of o of Pre selected Mfg Maximum of ports per sample Array Layout samples per array 6 por array 2 ii 1 quadruplicates 1 with Taq 6 15 ri 1 View Array Layout 1 View Array Layout 24 23 1 View Array Layout 2 31 View Array Layout ala 47 View Array Layout 63 1 4 View Array Layout View Array Layout LEN MO 4 View Array Layout View Array Layout g a 2 0 0 0 0 0 0 0 0 0 View Array Layout 9 Choose your assays and controls a Search for assays using one of the following options Notes Click Search by Keyword to display search options Enter a keyword select a category optional select additional search criteria for example species then click Search When the search results are returned select your assays click Add to Card then click Return to Configurator Click Search by Batch ID to display search options Enter or upload a batch of assay IDs optional select additional search criteria for example species then click Search When the search results are returned select your assays click Add to Card then click Return to Configurator Note You can place assays from more than one species on the same TagMan Array card Recommended for the TagMan Gene Sets Click Select a Gene Panel to display a suggested list of gene panels Sele
12. PCR products or used during sample preparation and clean gloves Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keepreactions and components capped as much as possible Use a positive displacement pipette or aerosol resistant pipette tips Clean lab benches and equipment periodically with 10 bleach solution TaqMan Array Micro Fluidic Cards User Guide 65 D Appendix D Prevent Contamination Notes 66 TaqMan Array Micro Fluidic Cards User Guide Numerics 7900HT Fast Real Time PCR System 3 A AIF See Assay Information File amplification no poor amplification 55 56 noisy amplification plot 54 analyze the TaqMan Array card 50 51 Applied Biosystems contacting vii Technical Support vii Array Information CD 26 Array Map File description 27 formats 27 Assay Information File description 27 B balance cards 39 41 biohazardous waste handling Xii bubbles 53 C CAUTION description x 48 cDNA convert from RNA 34 recommended amounts 36 centrifuge system balance cards 39 41 bucket requirements 39 centrifuge a TagMan Array card 39 components 5 EASY set control panel 40 QUI
13. RNA to cDNA Master Mix 200 reactions 4390778 High Capacity RNA to cDNA Kit 50 reactions 4387406 Notes 60 TaqMan Array Micro Fluidic Cards User Guide Appendix C Required Materials For Preparing the TaqMan Array Micro Fluidic Card For Preparing the TaqMan Array Micro Fluidic Card Reagents Item Size Description Part Number TaqMan Gene Expression Master Mix Mini Pack One 1 mL tube 4370048 2X 1 Pack One 5 mL bottle 4369016 2 Pack Two 5 mL bottles 4369514 5 Pack Five 5 mL bottles 4369510 10 Pack Ten 5 mL bottles 4369542 Bulk Pack One 50 mL bottle 4370074 TaqMan Universal PCR Master Mix 1 Pack One 5 mL bottle 4304437 with AmpErase UNG 2X 2 Pack Two 5 mL bottles 4364338 5 Pack Five 5 mL bottles 4364340 10 Pack Ten 5 mL bottles 4305719 Bulk Pack One 50 mL bottle 4326708 TagMan Universal PCR Master Mix No 1 Pack One 5 mL bottle 4324018 AmpErase UNG 2 Pack Two 5 mL bottles 4364341 5 Pack Five 5 mL bottles 4364343 10 Pack Ten 5 mL bottles 4324020 Bulk Pack One 50 mL bottle 4326614 TagMan Array Custom Micro Fluidic One TaqMan Array card See Chapter 2 Card 384 wells per card on page 9 for ordering TaqMan Gene Set One TaqMan Array card information 384 wells per card TaqMan Gene Signature Array The endogenous control Gene Signature Arrays come in sets of two cards the remaining Gene Signature Arrays come
14. Services Support Manage Shopping Baskets e eb Order History Reorder amp Welcome Laurie Swenson Not Laurie Swenson En Favorites N Promotions Quotes Workspace On the Store Log In page enter your user name and password Note If you are already logged in the Store Log In page does not appear Go to the next step Select the TagMan Array card to reorder then click at the top right of the page TaqMan Array Micro Fluidic Cards User Guide 21 Available Formats Chapter 2 Order the TaqMan Array Micro Fluidic Card Available Formats The table below provides a list of available TagMan Array Micro Fluidic Card formats for selected TagMan Gene Expression Assays Illustrations of each format follow Format Part No Se gen Me ius Sane ber Cd bar Lid Assays No Controls 8 per card 12 4342247 11 10 1 8 quadruplicates 1 16 4346798 15 10 1 8 triplicates 1 24 4342249 23 10 1 8 duplicates 1 32 4346799 31 10 1 4 triplicates 2 48 4342253 47 10 1 8 no replicates 1 64 4346800 63 10 1 2 triplicates 4 96a 4342259 95 10 1 4 no replicates 2 96b 4342261 95 10 1 2 duplicates 4 192 4346802 191 10 1 1 duplicates 8 384 4342265 380 10 1 4 replicates 1 no replicates 8 t After the minimum quantity has been fulfilled you can order TaqMan Array cards in increments of one Notes Format 12 11 target assays 1 mandatory manu
15. Short reaction time 0 5 to 1 hour High Capacity RNA to cDNA Kit Protocol High Capacity RNA to cDNA Master Mix A single tube of reagents streamlines the workflow and reduces experimental variability 5X master mix The RNA can be up to 80 of the final reaction volume High Capacity RNA to cDNA Master Mix Protocol High Capacity cDNA Reverse Transcription Kit Reaction components are individually available 4 tubes 10X RT Buffer 25X dNTP Mix 10X RT Random Primers MuLV Reverse Transcriptase High Capacity cDNA Reverse Transcription Kits Protocol IMPORTANT When performing reverse transcription Do not exceed the amounts of total RNA recommended in the protocol To avoid introducing a 3 bias and adversely affecting the TaqMan Array card results do not use any RT kits that use oligo d T primers exclusively Store the cDNA Samples If you will not be using the cDNA samples immediately store the samples at 15 to 25 C To minimize repeated freeze thaw cycles of cDNA Applied Biosystems recommends that you store your cDNA samples in aliquots Notes 34 TaqMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment This chapter covers E Prepare the TagMan Array Micro Fluidic Card a 36 B Run the TagMan Array Micro Fluidic Card 2 0 KK KK KK KK 46 B Analyze the Experiment mh 50 TaqMan Array Micro Fl
16. a guide N gue PHYSICAL INJURY HAZARD Take care when trimming off the fill strip Use scissors rather than razor blades or other unprotected cutting devices Notes TaqMan Array Micro Fluidic Cards User Guide 45 4 Chapter 4 Perform the Experiment Run the TaqMan Array Micro Fluidic Card Run the TaqMan Array Micro Fluidic Card In the SDS software set up the experiment SDS plate gt Perform the run on a 7900HT Fast System page 48 document using the SDS Setup File included on the Array Information CD this page Note When running the TagMan Array card see the product part numbers under For Running and Analyzing the TagMan Array Micro Fluidic Card on page 62 About Running After the TagMan Array cards have been loaded and sealed they are stable for at least Multiple Cards 64 hours Therefore for high throughput TaqMan Array card processing you can use the Automation Accessory For information on the Automation Accessory refer to the Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Set Up the SDS 1 Start SDS Software v2 1 or later Plate Document 2 Select File New 3 Complete the New Document dialog box a From the Assay dropdown menu select AAC RQ or Relative Quantification b From the Container dropdown menu select 384 Wells TaqMan Low Density Array c Complete the remaining fields as shown then click OK New Docu
17. are automatically deleted if they have not been used after 12 months Select filter criteria from Display pull down lists to see results Sort columns using header arrows Open a design by clicking on the name which returns you to the Configurator To Edit Copy or Export click one design s corre left then use the button on the right of the r Edit allows you to name rename and describe your file Exported files may only be uploaded if they are SNPlex encrypted files with the enc file extension using File Upload on the upper right Organize your files by creating Projects using the Project column Edit link To delete one or more designs check the corresponding box es and then click Delete sponding box at the i Indicates parent child relationship Parent Child Display ME DI All Project x Test 0 Configuration Pending 2009 01 26 Edit Order 12609 11 48 51 0 Copy Delete Previous Next In the Configurator review and order the TagMan Array card according to step 12 on page 15 or click Previous to make changes Reorder from your shopping basket 1 2 Notes Go to www appliedbiosystems com At the top of the Home page from the My Baskets Orders dropdown menu select Order History Reorder Applied RA AB Biosystems united states Store Log Out 34 my Account WP My Baskets Orders J Quick order View My Basket Products Applications 4 Technologies
18. at www cdc gov TaqMan Array Micro Fluidic Cards User Guide xiii Important Safety Information Biological Hazard Safety xiv TagMan Array Micro Fluidic Cards User Guide Chapter 1 Introduction This chapter covers B Product Overview M NGA LUPA GA AE NA GG GA gira PE AAO GU GD Gn GANG 2 E Instrument Overview a kak a kak RR IR RI hh hh eee 5 M User Guide Overview RR KK KK KK KK RR KK KK k8 6 TaqMan Array Micro Fluidic Cards User Guide 1 1 Chapter 1 Introduction Product Overview Product Overview About TaqMan Array Micro Fluidic Cards Notes TaqMan Array Micro Fluidic Cards are 384 well cards preloaded with TagMan Gene Expression Assays The TagMan Array cards allow you to measure gene expression using the comparative C AAC method of relative quantitation You can run 1 to 8 samples per card against 12 to 384 TaqMan Gene Expression Assay targets including controls Three types of TagMan Array cards are available from Applied Biosystems TaqMan Array Custom Micro Fluidic Card Customizable card you choose the assays to include in your card Select TaqMan Gene Expression Assays from Applied Biosystems collection of Inventoried assays The TagMan Array Custom Micro Fluidic Cards are manufactured when ordered TaqMan Gene Sets Customizable card you choose the assays to include in your card The TagMan Gene Sets are a collection of TagMan Gene Expression Assays that de
19. example Before running always prepare fresh master mix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open User Attention Two user attention words appear in Applied Biosystems user documentation Each word Words implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Examples of the user attention words appear below Note There is a fill port on the left arm of each fill reservoir it is the larger of the two holes IMPORTANT When performing reverse transcription do not exceed the amounts oftotal RNA recommended in the kit protocol TagMan Array Micro Fluidic Cards User Guide V Preface How to Obtain More Information Safety Alert Safety alert words also appear in user documentation For more information see Safety Words Alert Words on page x How to Obtain More Information Related In addition to this guide the ZaqMan Array Micro Fluidic Cards User Guide Documentation PN 4400263 the documents listed below are available from Applied Biosystems To obtain this and additional documentation see How to Obtain Support on page vii
20. kk kk kk kk kk kk k X B Chemical Safety Guidelines eee X B AboutMSDSS i ceo ore woe b eae bebes MC CAM oe den r xi B Chemical Waste Safety Guidelines WAK KK esee xii B Waste Disposal sere gx t rA Rr Re eR RR Xu Rx Rd ai xii E Biological Hazard Safety 0 kk kk KK KK KK KK KK KK KK KK KK KK KK KK KK KK KH xii TaqMan Array Micro Fluidic Cards User Guide ix Important Safety Information Safety Alert Words Safety Alert Words Definitions Chemical Hazard Warning Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N Ye Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices NITE Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N Deldi Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations rr CHEMICAL HAZARD Some of the chemicals used with Ap
21. waist high so that it can be easily used b Turn the sealer so that the front end the starting position shown below is closest to you and the back end is farthest from you In the correct position the arrows on the sealer are pointing away from you Carriage GR2171 Starting position c Place the sealer s carriage in its starting position IMPORTANT Never insert a TagMan Array card into the sealer if the carriage is not in its starting position The TagMan Array card will be irreparably damaged if the carriage is moved across it toward its starting position 2 Insert a TagMan Array card into the sealer a Orient the TaqMan Array card in the proper direction over the sealer s insert plate The card s fill reservoir end should be the end closest to the arrows etched in the base of the sealer b Line up the card s rear pin grooves foil side up to the stylus pins on the sealer Spring clips Stylus pins Spring clip Az P a GR2174 Notes TaqMan Array Micro Fluidic Cards User Guide 43 Notes Chapter 4 Perform the Experiment Prepare the TaqMan Array Micro Fluidic Card c Gently place the card on top of the insert plate and ensure that the front end of the card is held securely in place by the spring clips d Gently push the card until it is seated securely in the insert plate Note When properly seated the TaqMan Array card s foil surface should be level w
22. 2 Order the TagMan Array Micro Fluidic Card 2 Order the TaqMan Array Micro Fluidic Card Ordering Information Product Description Literature Support Related Products Configure Compatible Instruments Configuration Tools id Save This Design f Export Assay List amp Print Array Layout 5 Select a E o Format Choose Assays El 9 amp Controls B o Customize Array Layout optional v 3 Review amp Order 12 Review and order your TagMan Array card a Review the card details If needed click Previous to change the information b Enter the quantity of cards to order The minimum quantity you can order is 10 cards Note After the minimum quantity has been fulfilled you can order TagMan Array cards in increments of one c Select your basket from the dropdown menu ia 3 Review 8 Order Q previous Array Details Name Test Description Format Format 96a 12a Species Selected Homo sapiens Assigned Wells 95 Unique Genes 94 Empty Wells 0 Control Assays 18s rRNA s BE 9 7 ero Required Field 12c 12d Q Preis 1 2a Trademark Notes TaqMan Array Micro Fluidic Cards User Guide 15 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card d Click Add to Basket You can Repeat step 8 on page 11 through step 12 on page 15 to continue ordering TaqMan Array cards Click Shopping Basket to check out E Your or
23. Applied Bibsystems User Guide TaqMan Array Micro Fluidic Cards VYY YYY YY 00 O D 8 0 9 e 9 a DO 828888888 O O O O O O O O O 8 O e 8 02028828 8 ON BAN HG e e e e e 8 O H e O e H WWW 00 e e 6 HE HE E E BC e ie O e 8 e M O MW WWW 6 e282 amp e e e NN O NON e S 0 0 6 0 O O O e WWW e eae e e 8 O BA NO BN E E E 0 0 0 8 9 0 O OM e 8 8 BAN AN NB NN BOP 0 0 e 0 O O 6 M 222 HA HO DD BBS o 0 0 0 e e O O MW O 8 OM e NA amp NM NM E NA DO DM O ES SC eee ee NN NM e e ie O M e W6 maid User Guide Applied A Bibbystems TaqMan Array Micro Fluidic Cards Introduction Order the TaqMan Array Micro Fluidic Card Prepare Your Samples Perform the Experiment For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DI
24. Array Notes 16 TaqMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card 2 Order the TaqMan Array Micro Fluidic Card 6 Optional On the product page click the Product Description tab for more information about the assays and the card layout 7 Click the Ordering Information tab 8 Log in a Click Log In Note If you are already logged in the Log In prompt does not appear Go to the next step b On the Store Log In page enter your user name and password If you are not a registered user click Register Now then follow the prompts 7 ordering Information Product Description Literature Support The TagMan Human ABC Transporter Array contains assays for 50 human genes in a ion to 14 endogenous controls in a 384 well array 8a Please Log In to add products to your Shopping Basket Favorites configure a product or to view products available for purchase in your country Product Name Part Number Quantity Package TaqMan Human ABC 4378700 4 cards Transporter Array Note See user s manual or package insert for limited label license d trademark information For Research Use Only Not for use in diagnostics procedures Trademark 9 Order your TagMan Array card a Select the card b Select your basket from the dropdown menu Notes TaqMan Array Micro Fluidic Cards User Guide 17
25. Em EE eg RR Xd ERR RES A Rx RR a ED x Chemical Safety Guidelines eee x ABOULV SD SS AA AO Xi Chemical Waste Safety Guidelines llle xii Waste DISp0Sal a AA ac aka X044 cbe it A PAA AE xii Biological Hazard Safety 3 axa kk as a kl wa k an kn sara a lA lk a SK eee xiii Introduction Jk kk kk kk kk kK KIR KIR KI KIR kK kk kk kk kk kk a kk kk a 1 Product OVENVIEW re yak a dl kk a ana a e Rc ROO HEP d n ad RAT Pa KR A eae ad jar 2 Instrument Overview lk kk kk kk kk kk kk kk kK hh kk kk 5 User Guide Overview kk kk kk kk KK KK KK KK KK KK kk kk kk kk kk kk kk kK ee 6 Order the TaqMan Array Micro Fluidic Card 9 Order the TaqMan Array Micro Fluidic Card cece KK eee 10 Available Formats si errom ea iedh 44a j a ak aba a a ahh hh hr 22 Array Information CD kk kk kk kk kk kk kK kK KK KK KK KK KK KK kk kk kK kK kk kk kk lk kk k 26 Prepare Your Samples eelsees 31 Prepare Your Samples i kk kk kk kk kK kK kK kk KK KK KK kk kk kk kk kk kk kk kk kk kk ka 32 Store the cDNA Samples kk kk kk kk kk kK KK KK KK KK KK KK KK KK kk kk KK kk kk kk kk 34 Perform the Experiment 00 0 KK RR RR RR RR RR KK 35 Prepare the TagMan Array Micro Fluidic Card 0 0 36 Run the TagMan Array Micro Fluidic Card 0 kK cece eens 46 Analyze the Experiment i x sk sk xw ka k4 Ka a Kik n eee 50 TaqMa
26. Gene Expression Assays TaqMan Endogenous Controls TaqMan Gene Expression Assays 4 Click TaqMan Custom Array or TaqMan Gene Sets 384 Well Arrays Micro Fluidic Card TaqMan Custom Array TaqMan Gene Signature Arrays Human ABC Transporter Array Human Alzheimer s Array and Mouse Alzheimer s Arrav TaqMan Gene Sets Notes TaqMan Array Micro Fluidic Cards User Guide 19 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card 5 On the product page click the Ordering Information tab then click Configure Ordering Information Product Description Literature Support TaqMan Gene Sets are genes grouped by pathway or disease target class which can be ordered separately as a custom plate or configured directly onto TagMan Arrays that enable you to perform hundreds of real time PCR reactions simultaneously on the Applied Biosystems 7900HT Fast Real Time PCR System Simple to implement TaqMan Gene Sets are customizable and when on a TagMan Array require minimal amounts of sample allow for 1 to 8 samples to be run in parallel against multiple TaqMan Gene Expression Assay targets and don t require liquid handling robotics or complex pipetting How to Order Open the Ordering Guide 4 8 MB and follow the steps to add TaqMan Gene Sets to your shopping basket and check out Please Log In to add products to your Shopping Basket Favorites
27. If a large amount of air is released it can push the reaction mix out of the fill reservoir via the vent port or introduce bubbles into the fill reservoir 27 GR2159 38 TagMan Array Micro Fluidic Cards User Guide Section Centrifuge the After the fill reservoirs have been filled with the sample specific PCR reaction mix TagMan Array centrifuge the TagMan Array card to distribute the reaction mix to the reaction wells Card IMPORTANT You must use a Sorvall or Heraeus centrifuge with the Sorvall Heraeus Custom Buckets and card holders The Custom Buckets and card holders are custom made for the TaqMan Array cards Do not use any other centrifuge or bucket card holder system for this procedure For more information refer to the user manual for your centrifuge Note The Sorvall and Heraeus centrifuges have either a touchpad control EASYSet models or knob operated control panel QUIKSet models Both centrifuges use the same bucket type and card holder 1 Place TagMan Array cards into the Sorvall Heraeus buckets a Obtain an empty Sorvall Heraeus Custom Bucket and card holder Note The centrifuge holds four Sorvall Heraeus buckets Each bucket holds up to three TaqMan Array cards loaded and or blank balance cards in the card holder The card holder supports the TagMan Array card during centrifugation b Place the bucket on a lab bench with the label facing you c Insert TagMan Array cards into
28. Kset control panel 40 TaqMan Array card placing in bucket 39 chemical safety X chemical waste safety xii condensation 53 contamination See prevent contamination 65 conventions for describing menu commands V TaqMan Array Micro Fluidic Cards User Guide Index IMPORTANTS v Notes V text V user attention words V D DANGER description X downstream analysis 51 E EASY set control panel 40 efficiency TagMan Gene Expression Assay 3 excess reaction mix 54 experiment analyze 50 51 before you begin 5 review results 50 F filling a TagMan Array card guidelines 37 procedure 38 formats Array Map File 27 TaqMan Array card 22 25 G genomic DNA detection 33 guidelines chemical safety X chemical waste safety xil filling a TaqMan Array card 37 prevent contamination 65 RNA template 32 H Help system accessing Vil import the SDS Setup File 46 IMPORTANT description X instruments 67 Index required components 3 required to run TaqMan Array card 5 L laboratory practices 65 M materials See required materials 59 63 MSDSs description Xi obtaining Vii xi multiple cards 46 O online Help See Help system ordering TaqMan Array Custom Micro Fluidic Card 10 19 21 TagMan Gene Sets 10 19 21 TaqMan Gene Signature Array 16 P PCR step TaqMan Array card placing in bucket 39 poor precision 55 prevent contamination good laboratory practices for PCR 65 guidelines 65 product
29. Man Array Micro Fluidic Cards User Guide 49 Chapter 4 Perform the Experiment Analyze the Experiment Analyze the Experiment Review the results this page v Optional Perform downstream analysis page 51 Notes Review the Results Note When analyzing the experiment see the product part numbers under For Running and Analyzing the TagMan Array Micro Fluidic Card on page 62 The comparative Cy AAC method uses arithmetic formulas to determine change in expression of a target in an experimental sample relative to the same target in a reference sample The AAC method is used for high throughput measurements of relative gene expression when there are many genes in many samples For details on how to analyze AAC experiments and set up an RQ Study refer to the 7900HT Fast Real Time PCR System Relative Quantitation Using Comparative Cy Getting Started Guide Review the chapter on analyzing and viewing RQ Study data in the RQ Manager Brief procedures are provided below 1 Transfer the SDS plate document file sds into an RQ Study then analyze the study For optimal results Applied Biosystems recommends the following For Applied Biosystems TaqMan master mixes analyze the study with Automatic Baseline and Manual C set to 0 2 View the amplification plot then review the baseline and threshold settings If needed adjust the baseline and threshold settings for individual assays IMPORTANT
30. SCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED LICENSE A license to perform the patented 5 Nuclease Process for research is obtained by the purchase of i both Licensed Probe and Authorized 5 Nuclease Core Kit ii a Licensed 5 Nuclease Kit or iii license rights from Applied Biosystems The TagMan Array Micro Fluidic Card contains Licensed Probe Use of this product is covered by US patent claims and corresponding patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research The right to use this product in the 5 Nuclease Process under the applicable claims of US patents and corresponding patent claims outside the United States can be obtained through purchase of an Authorized 5 Nuclease Core Kit Except under separate license rights available from Applied Biosystems no right under any other patent claim or to perform comme
31. Software v2 1 or later Fast Real Time PCR System Note The SDS software includes a relative quantitation program Depending on the SDS software version it may be called RQ Manager or AAC Study 7900HT TaqMan Array Micro Fluidic Card Upgrade hardware upgrade kit e TagMan Array Micro Fluidic Card Thermal Cycling Block e TaqMan Array Micro Fluidic Card Sealer Four centrifuge buckets and card holders specific to the Sorvall or Heraeus centrifuge e 7900HT TagMan Array Micro Fluidic Card Chemical Installation Kit Spectral Calibration Kit TagMan Array Micro Fluidic Card Instrument Verification RNase P Kit Calibration Cards 4 cards Before You Begin Before you perform your quantitation experiment make sure that e Notes Within the last 6 months a background run pure dye runs and an instrument performance run RNase P run have been performed on the 7900HT Fast System with the TaqMan Array Micro Fluidic Card Thermal Cycling Block installed For more information refer to the SDS Online Help or Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide You are familiar with the safety guidelines in the Safety and EMC Compliance Information section of the Applied Biosystems 7900HT Fast Real Time PCR System Site Preparation Guide You are familiar with the safety guidelines related to use of your centrifuge as provided by the centrifuge manufacturer
32. T HsQ0742749 si FAM Table Settings None zi E EOMES Hs00172872 m1 FAM Ha Posit Flag Sample Task Fos Hmo ime EW amp Fere Bor EE E rr FLTI Hs00176573 m1 FAM laz SampleDi ACTC Hs0806316_m1 Target j Sr FN1 Hs00277509_m1 FAM rm Aa Sample01 AFF HsO0173490 mi Target T FOXA2 Hs00232764_mi FAM rg Sample01 BRDCHsQU217848 m1 Target C FOXDS HsQ0255287 si FAM c JA Sample01 C034 H500156373 m1 Target r GABRES HsOO24 1458 mi FAM AB SampleD1 CD9 Hs00233521 m1 Target r GAL Hs00544355 m1 FAM I A ISample01 CDH5 Hs00174344 m1 Target r GAPD Hs99999905 m1 FAM AB Sample01 CD 2 Hs00230919 m1 Target F GATAA Hs00171403 m FAM CJ Ng Sample01 CGB H500361224 gH Target T GATAS Hel0232016 m1 FAM r a10 Sample01 COLTA1 Hs00164004 mi Target F GBYQ Hs00230965 Th FAM AT Sample01 85 Hs99999901 si Endogeno T GCG Hs00174967 m FAM A12 Sample01 COLZA1 Hs00156563 mi Target r Hs00172692 mi FAM a lAI3 Sample01 COMMD3 Hs00201350_m1 Target r oe FAM lata Sample01 CRABP2 Hs00275636 mi Target r GFAP Hs00157674 m1 FAM AIS Sample CTNNB1 Hs00170025 mi Target E GRR7 HaNn917999 n1 FAM IP A16 Sample01 DDX4 Hs00251859 mi Target z A17 Sample01 DES Hs00157258 mi Target Add Detector Oca a paluan laia Sample01 DNMT3B Hs0017 1876_m1 Target T A19 Sample01 EBAF Hs00745761 si Target ji Passive Reference Rox 4 x j zk gt T Omit Well s Notes
33. agMa Gene Sets are customizable and when on a TaqMan Array require minimal amounts of sample allow for 1 to 8 samples to be run in parallel against multiple TaqMan Gene Expression Assay targets and don t require liquid handling robotics or complex pipetting How to Order Open the Ordering Guide 4 8 MB and follow the steps to add TaqMan Gene Sets to your shopping basket and check out Please Log In to add products to your Shopping Basket Favorites configure a product or to view products available for purcha i your country O Product Name Part Number Quantity Package TaqMan Array LDA 1 cards configure 7 On the Store Log In page enter your user name and password If you are not a registered user click Register Now then follow the prompts Note If you are already logged in the Store Log In page does not appear Go to the next step 8 Select a format for your TagMan Array card a Enter a name and description for your card You cannot enter more than 24 characters for the card name b Select a format For more information see Available Formats on page 22 c Click Next Notes TaqMan Array Micro Fluidic Cards User Guide 11 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card 8a 8b 1 Select a Format e tation of a particular array wnload the All Array Maps pdf 57Kb ui ra nt
34. ast System detects the real time amplification of your targets then determines relative levels of gene expression from the fluorescence data that are generated during PCR Fill reservoir 1 of 8 A reservoir for the sample before it is centrifugally transferred into the wells See below for an enlarged view of the fill reservoir L_ Fill strip The segment of the TaqMan Array card that contains the eight fill reservoirs After the card is centrifuged and sealed you trim off the fill strip Barcode For each TaqMan Array card a unique identifier that can be scanned into database systems for tracking The fill reservoir includes a fill port on the left and a vent port on the right Use the fill port to add reaction mix to the TaqMan Array card Fill port Vent port GR2158 TagMan Array Micro Fluidic Cards User Guide Chapter 1 Introduction 1 Instrument Overview Instrument Overview The TagMan Array cards must be e e Centrifuged with a Sorvall or Heraeus centrifuge Sealed with a TaqMan Array Micro Fluidic Card Sealer then trimmed Run on an Applied Biosystems 7900HT Fast Real Time PCR System The instrument must have a TagMan Array Micro Fluidic Card Thermal Cycling Block installed Required Components Instrument Component Sorvall or Heraeus Sorvall Heraeus Custom Buckets centrifuge Card holders Applied Biosystems 7900HT Sequence Detection Systems SDS
35. aste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require s
36. atically stops after 1 min per the programmed sequence START button Ei b Repeat step a so that the TaqMan Array cards are centrifuged for a total of two consecutive 1 minute spins IMPORTANT To ensure complete distribution of the PCR reaction mix you must centrifuge the TaqMan Array cards for a total of two consecutive l minute spins 5 Remove the TaqMan Array cards a Press the Open button b When the cover has fully opened remove the buckets from the centrifuge then remove the card holders from the buckets c Remove all TaqMan Array cards from the buckets by gently lifting them by their carrier sides 6 Examine the TaqMan Array cards to be sure filling is complete The amount of PCR reaction mix remaining in the fill reservoirs should be consistent from reservoir to reservoir The TaqMan Array Micro Fluidic Card Sealer isolates the wells of a TaqMan Array card after it is loaded with PCR reaction mix The sealer uses a precision stylus assembly carriage to seal the main fluid distribution channels of the TagMan Array card IMPORTANT Proper operation of the sealer using a slow steady and deliberate motion is critical to the successful use of the TaqMan Array card 42 TagMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Prepare the TagMan Array Micro Fluidic Card 1 Position the sealer a Place the sealer on a sturdy lab bench approximately
37. ation you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 MSDSs hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then click MSDS Search 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer TaqMan Array Micro Fluidic Cards User Guide xi Important Safety Information Chemical Waste Safety Guidelines Chemical Waste Safety Guidelines Waste Disposal xii To minimize the hazards of chemical waste e Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary w
38. bar code number on the label of each shipped rack or plate N A Plate ID that consists of the shipment ID plus a unique numeric suffix that identifies the rack or plate containing the assay Vial Tube Type The type of vial or tube that contains the assay N A Vial Tube ID A unique 10 digit bar code number on the bottom of each assay N A vial or tube that identifies it Well Location on the The location of the assay on the associated shipping rack or A1 Shipping Rack or Plate plate Assay Mix The concentration of the assay including both primers and 2X Concentration probe Forward Primer Name The name of the forward primer assigned by the design N A software that consists of the assay ID plus an F suffix Forward Primer The nucleotide sequence of the forward primer N A Sequence Forward Primer The concentration of the forward primer uM 18 Concentration Reverse Primer Name The name of the reverse primer assigned by the design N A software that consists of the assay ID plus an R suffix Reverse Primer The nucleotide sequence of the reverse primer N A Sequence Reverse Primer The concentration of the reverse primer uM 18 Concentration Notes 28 TagMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card Array Information CD Field name Description of content Example Reporter 1 Name The name of the reporter 1 oligonucleotid
39. binding protein Hs99999910 m1 Mm00446973 m1 Rn01455648 m1 TFRC Transferrin receptor Hs99999911 m1 Mm00441941 m1 Rn01474695 m1 UBC Ubiquitin C Hs00824723 m1 Mm01201237 m1 Rn01789812 g1 YWHAZ Tyrosine 3 monooxygenase or tryptophan 5 monooxygenase activation protein zeta polypeptide Hs00237047 m1 Mm01158417 g1 Rn00755072 m1 TaqMan Array Micro Fluidic Cards User Guide 57 EV Appendix B TaqMan Endogenous Control Assays Notes 58 TaqMan Array Micro Fluidic Cards User Guide Appendix C Required Materials This appendix covers E For Preparing Your Samples k kk kk KK KK KK KK KK KK KK KK KK KK KK KK 60 E For Preparing the TagMan Array Micro Fluidic Card 0 005 61 E For Running and Analyzing the TaqMan Array Micro Fluidic Card 62 B ForGeneral Use 2 sus oa la GT RU ted pex dora ke Bea Av n ADU Reds 63 TaqMan Array Micro Fluidic Cards User Guide 59 Appendix C Required Materials For Preparing Your Samples For Preparing Your Samples Reagents Item Size Part Number TaqMan Gene Expression Cells to CT 100 lysis reactions 500 PCR AM1728 Kit 400 lysis reactions 2000 PCR AM1729 High Capacity cDNA Reverse 200 reactions 4368814 Transcription Kit 200 reactions with RNase 4374966 Inhibitor 1000 reactions 4368813 1000 reactions with RNase 4374967 Inhibitor High Capacity
40. ct your gene panels click Add panels to Card then click Return to Configurator Note For more gene panels go to www appliedbiosystems com tools workflow Choose the endogenous controls for your experiment using any of the search options described in step a above Note Applied Biosystems recommends that you use the TaqMan Endogenous Control Assays to select your endogenous controls For more information see Appendix B on page 57 and refer to the Applied Biosystems Application Note Using TagMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies 12 TagMan Array Micro Fluidic Cards User Guide Section 2 c Review the gene symbols added to your TaqMan Array card If needed you can Repeat step a above to add more assays panels to the card Select a gene symbol then click Remove to remove assays panels from the card d In the Manufacturing Controls box select a gene symbol from the dropdown menu Note The well locations for the controls are predetermined based on the TagMan Array card format you selected in step 8 on page 11 e Click Next 9c e Previous Next o 3 ways to search for and Gene Symbol Assay ID a Array Details choose assays T BMP10 Hs00205566 m1 Name Test Search by Keyword D BMPR2 Hs00176148 m1 Format Format 96a T GDF10 Hs00192033 m1 Assigned Wells 95 T SMAD1 Hs00195432 mi Empty Wells 0 D SMAD9 Hs00195441 m1 Unique Gen
41. der was successfully added to your Shopping Basket Applied Biosystems ships the TaqMan Array cards at ambient temperature Upon receipt store the cards at 2 to 8 9C The cards are stable for up to 2 years from the manufacturing date Order TaqMan 1 Goto www appliedbiosystems com Gene Signature Arrays 2 In the I Want to Buy box click TaqMan Gene Expression Assays Plates amp Arrays I Want to Buy TaqMan Gene Expression Assays Plates amp Arrays TaqMan MicroRNA Assays TaqMan SNP Genotyping Assays Real Time PCR Reagents amp Kits Custom Primers amp Probes Assays amp Custom Products mahila 3 Under Arrays click 384 Well Arrays Micro Fluidic Card Gene Expression Assays 8 Arrays Which gene expression assay is right for you Read our TaqMan Gene Expression Assays Product Guide PDF 1 0 MB Arrays TaqMan Custom 96 Well Arrays Plating Service Microplate 38 Vell Arrays Micro Fluidic Card Individual Assays Custom TaqMan Gene Expression Assays TaqMan Endogenous Controls TaqMan Gene Expression Assays 4 Click TaqMan Gene Signature Arrays 384 Well Arrays Micro Fluidic Card TaqMan Custom TaqMan Gene Array Signature Arrays Human ABC Transporter TaqMan Gene Sets Array Human Alzheimer s Array and Mouse Alzheimer s Arrav 5 On the TagMan Gene Signature Arrays page select the appropriate Gene Signature
42. ds User Guide workflow for performing experiments with 6 TaqMan Array Custom Micro Fluidic Card ordering 10 19 21 product overview 2 TaqMan Array Micro Fluidic Card Sealer 5 42 diagram 43 TaqMan Array Micro Fluidic Card Thermal Cycling Block 5 TaqMan Gene Expression Assay efficiency 3 product overview 3 TagMan Gene Sets ordering 10 19 21 product overview 2 TaqMan Gene Signature Array ordering 16 product overview 2 Technical Support contacting vii training information on vii troubleshooting 54 bubbles 53 condensation 53 drained fill reservoir 54 no poor amplification 55 56 noisy amplification plot 54 poor precision 55 reaction mix leaks 53 too little reaction mix 53 U user attention words described V W WARNING description X waste disposal guidelines Xii workflow 6 TaqMan Array Micro Fluidic Cards User Guide Index 69 Index 70 TagMan Array Micro Fluidic Cards User Guide www appliedbiosystems com Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 07 2010
43. e cDNA samples on ice Resuspend the cDNA samples by inverting the tube then gently vortexing Mix the master mix thoroughly by swirling the bottle For each sample label a 1 5 mL microcentrifuge tube then add the required components to the labeled tube Cap the microcentrifuge tubes then gently vortex the tubes to thoroughly mix the solution Briefly centrifuge the tubes to spin down the contents and eliminate air bubbles Fill the TaqMan Guidelines to ensure optimal PCR performance Array Card e Notes Do not remove a TaqMan Array card from its packaging until the packaging has reached room temperature and you are ready to fill it with sample specific PCR mix Prolonged exposure to indoor lighting can photo degrade the fluorescent probes contained within the card Do not expose the card to sunlight Fill each fill reservoir with sample specific PCR mix made from a single cDNA sample Add 100 uL of the sample specific PCR mix per fill reservoir to ensure adequate filling Volumes smaller than 100 uL will result in insufficiently filled cards Do not add sample after centrifuging the cards When you centrifuge the card the sample specific PCR mix resuspends the dried TaqMan probes and primers within the wells of the card Adding sample after centrifuging disrupts the resuspended assay positions To ensure a high degree of reproducibility Applied Biosystems recommends scheduling your runs so that each card is
44. e document for your TagMan Array card in the SDS software Applied Biosystems recommends that you import the SDS Setup File see step 4 under Set Up the SDS Plate Document on page 46 The SDS software uses the information in the SDS Setup File to configure the plate grid and setup table in the SDS plate document The figure below shows an SDS plate document after an SDS Setup File has been imported IMPORTANT Modifying the contents of the SDS Setup File can corrupt the file making the file unusable that is you will not be able to access information for the TaqMan Array card l gl xi 8 19 20 2 3 24 Setup instrument mu LIPS was at aj sameie Name Sample01 Use Detector Reporter Task T FAM The SDS aa E FAM an ft u u u u u u r FAM software nm mm r pue CAM displays well JIH a mi FAM u u u u u n r CDH5 Hs00174344 mi FAM information if Agr a o FAM r CGB Hs 0361224 gH FAM 5 r COLIAT Hs00164004_mi FAM you leave the i COL2ZAI Hs001S6565_mi FAM LANH COMMD3 Hs00201350 mi FAM cursor over ene soni ronae m FAN LEE n u n n n n n mm mn n mn m DDX4 Hs00251659 mi FAM cm the well T DES Het0157258 mi FAM gt TA r DNMT3B Hs00171876 mi FAM t AY r EBAF HsDO745761 s1 FAM T EEFTA
45. e probe assigned by N A the design software that consists of the assay ID and a suffix code M1 or M2 The letter in the suffix code identifies the reporter dye that is covalently bound to the fluorogenic probe The number identifies the DNA strand used to design the probe e 1 Forward strand design e 2 Reverse strand design For example in the name KR14TD A22TM1 the letter M indicates that the probe is labeled with the FAM dye and the number 1 indicates that the probe was designed to the forward strand Reporter 1 Dye The reporter dye label for the reporter 1 probe FAMTM Reporter 1 Sequence The nucleotide sequence of the reporter 1 probe N A Reporter 1 The concentration of the reporter 1 probe uM 5 Concentration Reporter 1 Quencher The quencher used for reporter 1 probe for example Minor NFQ Groove Binder Non Fluorescing Quencher MGB NFQ Reporter 2 Name Not applicable to TagMan Gene Expression Assay N A Reporter 2 Dye Reporter 2 Sequence Reporter 2 Concentration Reporter 2 Quencher Context Sequence The 25 nucleotide sequence surrounding the probe including AGGAAACGTCTGGGCGAT the targeted exon s GACAACC Design Strand Indicates the strand used to design the probe N A Forward The probe binds to the same strand as the forward primer Reverse The probe binds to the same strand as the reverse primer Category The Celera Panther Protein Classification Level 1 for the gene Nucl
46. eic acid binding Category ID A unique 10 character alphanumeric abbreviation of the NUC 1090000 Panther category classification for the assay Group The Celera Panther Protein Classification Level 2 for the gene Replication origin binding protein Group ID A unique 10 character alphanumeric abbreviation of the 1090300000 Panther group classification for the assay Gene Symbol The Entrez Gene symbol for the gene CDC6 Gene Name The Entrez Gene name for the gene Cell division cycle 6 homolog S cerevisiae Chromosome The chromosome containing the gene 17 Species The organism for which the assay was designed Homo_sapiens Target Exons The public accession number s of the exon s that are spanned by the probe 2 Notes TaqMan Array Micro Fluidic Cards User Guide 29 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Array Information CD Field name Description of content Example NCBI Gene Reference The NCBI transcript identification number that corresponds to NM 001254 3 U77949 1 the gene AF022109 1 CR598029 1 BC025232 1 NCBI SNP Reference Not applicable to TagMan Gene Expression Assays N A Medline Reference PubMed references for the gene N A Celera ID The unique Celera Discovery System CDS assay identification hCT1643963 2 number for the gene hCT2294847 0 Cytogenetic Band The chromosomal band where the gene is located If 17g21 3 unavailable then the chromosome number is provided
47. es 94 D sp1 Hs00293689 s1 Manufacturing Controls D TGFBR2 Hs00559661 m1 Gene Symbol Location PF ACYR1 Hs00153836 m1 185 RNA wla 9d I LEFTY2 Hs00745761 s1 F TGFBi Hs99999918 mi x ml GENOME E Select a Gene Panel e defined onding to a r target class Remove 9C Previous Next 6 9e 10 Optional Customize the TaqMan Array card layout a To change the location of the assays in the card use the up down arrows or enter the desired well location Note If you change the well location for one replicate all other replicates are moved accordingly Note The well locations for the controls are fixed based on the TaqMan Array card format you selected in step 8 on page 11 Notes TaqMan Array Micro Fluidic Cards User Guide 13 2 Chapter 2 Order the TagMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card b Click Update Array Layout Note To return to the previously saved layout click Reset Layout c Click Next You can change the location of assays using the up and down arrows in the Move column Note that the locations for manufacturing controls are fixed Skip this Step Q Previous Next Ez Location Gene Symbol Assay ID Array Details v AO1 BMP10 Hs00205566 m1 pame Test a Format Format 96a v A02 BMPR2 Hs00176148 m1 Assigned Wells 95 AO3 GDF10 Hs00192033 m1 Empty Wells o 10a Unique Genes 94 e A04 SMAD1 Hs00195432 mi p Manufacturing Cont
48. ession TagMan Array Micro Fluidic Cards User Guide Chapter 1 Introduction 1 Product Overview About TaqMan TaqMan Gene Expression Assays are a comprehensive collection of Inventoried and Gene Expression Made to Order Assays probe and primer sets for performing quantitation experiments Assays on genes from several species Note Only Inventoried Assays can be placed onto a TagMan Array card If you require a Made to Order Assay please contact your local Applied Biosystems Sales Representative The TaqMan Gene Expression Assays Use TaqMan reagents 5 nuclease chemistry to amplify and detect the target in cDNA samples When possible can amplify target cDNA without amplifying genomic DNA The suffix of the assay ID indicates the assay placement and thus genomic DNA detection see Detection of genomic DNA During Amplification on page 33 e Consist of two unlabeled primers and one 6 FAM dye labeled TagMan MGB probe The TaqMan Gene Expression Assays are Designed with an automated design system Designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions Preformulated and ready to use Efficiency of TagMan Gene Expression Assays A statistically significant number of TaqMan Gene Expression Assays have been tested for PCR efficiency When tested across a 6 log range all assays approached 10096 efficiency 41096 Refer to the Appl
49. facturing control 8 unique samples Replicates Aa eG Aa oag Cq Ma ua ria c r o mimio oj m ziz 22 23 24 22 TagMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card 2 Available Formats Format 16 15 target assays 1 mandatory manufacturing control 8 unique samples Replicates Port A 1 5 1 2 62 D E 3 3 G 4 4 5 Di 5 K 6 i 6 M ra 7 O 8 NAT 1 2 3 4 5 6 HA 8 9 10 11 12 13 14 15 16 7 18 19 20 21 22 23 24 Format 24 23 target assays 1 mandatory manufacturing control 8 unique samples Replicates Port A 1 Bi 1 2 CAN c 2 1 D iz JIE 3 H3 G 4 m 4 P 5 i n 5 1 K 6 i 6 1 M 7A 7 N Y o 8 2 lt lt 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Format 32 31 target assays 1 mandatory manufacturing control 4 unique samples Replicates Port lt a 1 1 c 2 D 2 1 G 4 r lt 5 3 1 K 6 L Ma 4 5 z 3 Notes TaqMan Array Micro Fluidic Cards User Guide 23 2 Chapter 2 Order the TaqMan Array Micro Fluidic Card Available Forma
50. fine specific target classes pathways or diseases for example the TagMan Array Human Apoptosis Micro Fluidic Card Select assays from the TagMan Gene Sets then customize the card according to your research needs TagMan Array cards with TaqMan Gene Sets are manufactured when ordered Note You can substitute assays within a TagMan Gene Set with TaqMan Gene Expression Assays from Applied Biosystems collection of Inventoried assays TaqMan Gene Signature Array Preconfigured card The TaqMan Gene Signature Arrays contain preselected TagMan Gene Expression Assays that target specific gene classes for example Human Stem Cell Pluripotency markers The TaqMan Gene Signature Arrays are manufactured and placed in inventory A complete list of the TagMan Gene Expression Assays that are available for each type of TaqMan Array card can be found on the Applied Biosystems web site For ordering information see Chapter 2 on page 9 Key Features Small volume design minimizes sample and reagent consumption Streamlined reaction setup saves time and reduces labor intensive steps Provides access to high throughput 384 well format without liquid handling robotics Detects two fold discrimination at the 99 7 confidence level Provides standardization across multiple samples in multiple laboratories For More Information For more information on relative quantitation refer to User Bulletin 2 Relative Quantitation of Gene Expr
51. hrough v2 2 2 include the Contact your local Applied Biosystems Sales AAC Study program SDS Software v2 3 includes the Representative RQ Manager program Both programs are provided for relative quantitation analysis Real Time StatMiner Software Go to www integromics com StatMiner php 62 TaqMan Array Micro Fluidic Cards User Guide Appendix C Required Materials For General Use For General Use For more product recommendations visit the real time PCR decision tree www appliedbiosystems com qpcrtree Item Source Disposable gloves MLS Microcentrifuge MLS Microcentrifuge tubes 1 5 mL MLS Nuclease free Water not DEPC treated Applied Biosystems PN AM9930 Pipette tips with filter plugs MLS Pipettes positive displacement or air displacement MLS Polypropylene tubes MLS Tris EDTA TE buffer pH 8 0 MLS Vortexer MLS Notes TaqMan Array Micro Fluidic Cards User Guide 63 Appendix C Required Materials For General Use Notes 64 TagMan Array Micro Fluidic Cards User Guide Appendix D Prevent Contamination PCR assays require special laboratory practices to avoid false positive amplifications The high throughput and repetition of these assays can lead to amplification of one DNA molecule Good Laboratory When preparing samples for PCR amplification Practices for PCR Wear a clean lab coat not previously worn while handling amplified
52. ication Note Amplification Efficiency of TaqMan Gene Expression Assays for validation information Note Applied Biosystems does not recommend that you test assay efficiency on the TaqMan Array cards In order to calculate the efficiency correctly a dilution series with a minimum of 5 orders of magnitude must be used With cDNA samples in most cases it is not possible to obtain results over such a large dynamic range potentially leading to inaccurate results Notes TaqMan Array Micro Fluidic Cards User Guide 3 1 Chapter 1 Introduction Product Overview Applications The TaqMan Array cards are recommended for use TaqMan Array Micro Fluidic Card Diagram Notes In relative quantitation experiments using the comparative C4 AAC method With singleplex PCR A single primer probe set is present in each well of the TagMan Array card Only one target can be amplified per well In 2 step RT PCR In the reverse transcription RT step you convert total RNA to cDNA To perform reverse transcription Applied Biosystems recommends the High Capacity cDNA Reverse Transcription Kit or the High Capacity RNA to cDNA Kit nthe PCR step you combine your cDNA samples and a TagMan master mix add this reaction mix to the TagMan Array card then run the card on a 7900HT Fast Real Time PCR System The instrument must have a TagMan Array Micro Fluidic Card Thermal Cycling Block installed During PCR the 7900HT F
53. ics of interest in the Help system you can Review the table of contents Search for a specific topic Search an alphabetized index Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is only for submitting comments and suggestions relating to documentation To order documents download PDF files or for help with a technical question go to www appliedbiosystems com then click the link for Support See How to Obtain Support below How to Obtain Support For the latest services and support information for all locations go to www appliedbiosystems com then click the link for Support At the Support page you can Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches TaqMan Array Micro Fluidic Cards User Guide vii Preface How to Obtain Support viii TagMan Array Micro Fluidic Cards User Guide Important Safety Information This section covers B Safety Alert Words 2 2220 KK KK KK E KK KK KK
54. in 2x1 min Notes 40 TagMan Array Micro Fluidic Cards User Guide EASYSet control panel Buttons 1 4 are programming buttons QUIKSet control panel Acceleration settings Chapter 4 Perform the Experiment 4 Prepare the TagMan Array Micro Fluidic Card rpm Scroll indicator control Down ramp rate control controls controls Up ramp rate control Bucket type rpm Adjustment Adjustment indicator knob speed knob time number Mode settings 3 Place the buckets into the centrifuge a Press the Open button on the centrifuge to open the centrifuge cover Open cover button b Place each loaded bucket onto an open rotor arm of the centrifuge Make sure each bucket can swing easily within its slotted position on the rotor arm IMPORTANT The manufacturer recommends running the centrifuge with all four buckets If the buckets are not fully loaded with TagMan Array cards containing the sample specific PCR reaction mix place blank balance cards and card holders into the buckets Make sure the buckets and their contents are balanced opposing buckets should have matching weights Notes TaqMan Array Micro Fluidic Cards User Guide 41 4 Chapter 4 Perform the Experiment Prepare the TaqMan Array Micro Fluidic Card Seal the TaqMan Array Card Notes c Close the centrifuge cover 4 Run the centrifuge a Press the Start button The centrifuge starts then autom
55. in sets of four cards 384 wells per card Equipment Item Part Number Micropipettes 100 uL Major laboratory supplier MLS Sorvall or Heraeus centrifuge Contact your local Applied Biosystems Sales Rotor Representative Notes TaqMan Array Micro Fluidic Cards User Guide 61 Appendix C Required Materials For Running and Analyzing the TagMan Array Micro Fluidic Card Item Sorvall Heraeus Custom Buckets Sorvall Heraeus card holders TaqMan Array Micro Fluidic Card Sealer Part Number Included in the 7900HT TagMan Array Micro Fluidic Card Upgrade hardware upgrade kit For Running and Analyzing the TaqMan Array Micro Fluidic Card Instrument and Notes Accessories Item Part Number Applied Biosystems 7900HT Fast Real Time PCR System Contact your local Applied Biosystems Sales Representative 7900HT TaqMan Array Micro Fluidic Card Upgrade 4329012 hardware upgrade kit This kit includes e TaqMan Array Micro Fluidic Card Thermal Cycling Block e TaqMan Array Micro Fluidic Card Sealer Four centrifuge buckets and card holders specific to the Sorvall or Heraeus centrifuge e 7900HT TaqMan Array Micro Fluidic Card Chemical Installation Kit Spectral Calibration Kit TagMan Array Micro Fluidic Card Instrument Verification RNase P Kit Calibration Cards 4 cards Software Item Part Number SDS Software v2 1 or later Note SDS Software v2 1 t
56. ing techniques to avoid introducing bubbles when loading the fill reservoirs Adjust the baseline and threshold settings then reanalyze Remove the replicate well s as an outlier Leaky well s due to improper staking The QC Flag is BPR Omit the leaky well s from analysis then reanalyze the data Use a steady even motion when using the staker Not all of the card holder positions were filled before centrifuging Be sure to fill all of the card holder positions with blank TagMan Array cards before centrifuging Non validated centrifuge Use a Sorvall or Heraeus centrifuge Non AB master mix Use AB master mix No amplification or poor precision across many assays Non AB master mix No reagent master mix No sample Inhibitor in the sample Check the protocol be sure to add the correct amount and the correct type of sample and master mix Notes 56 TagMan Array Micro Fluidic Cards User Guide Table 1 Assays for normalization Appendix To help with normalization you can select a human mouse or rat endogenous control assay from the list of inventoried endogenous controls in Table 1 below experimental conditions and is sufficiently abundant across all tissues and cell types studied Note Applied Biosystems recommends that you experimentally validate all candidate genes to be used as endogenous controls Candidate endogenous contr
57. ith the base of the sealer The four spring clips ensure that the card is held in the proper position 3 Push the carriage across the base of the sealer in the direction of the arrows Use a slow steady and deliberate motion to push the carriage across the entire length of the TagMan Array card until the carriage reaches the mechanical stops It is important to avoid moving the carriage rapidly across the card N ezl t Ng The sealer has mechanical stops at both ends to prevent the carriage from coming off Therefore do not use excessive force or speed when pushing the carriage End position Move in this direction d IMPORTANT Do not move the carriage back before removing the TagMan Array card Remove the sealed TaqMan Array card by grasping its sides and lifting it off the sealer s insert plate In the middle of the sealer s insert plate there is a thumb slot to help you easily access one side of the card Thumb slot 44 TagMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Prepare the TaqMan Array Micro Fluidic Card 5 Inspect the TagMan Array card for proper sealing The indentations from the stylus assembly should match up with the card s main channels If the indentations do not match up or if the foil is in any way damaged do not use the TaqMan Array card 6 Using scissors trim the fill strip from the TagMan Array card Use the edge of the card s carrier as
58. ment The Assay field selections vary depending on your Assay AACt RO if software version Container a84 Wells Taqman Low Density Array Template Blank Template X Browse Barcode Recommended Click the Barcode field then scan or 3 Save Settings As My Default Cancel type the barcode 4 Import the SDS Setup File txt into the new SDS plate document a In the CD drive insert the Array Information CD that shipped with your TaqMan Array card b In the SDS software select File Import Notes 46 TaqMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Run the TaqMan Array Micro Fluidic Card c In the Import dialog box navigate to the SDS Setup File for your TagMan Array card then click Import The SDS software imports information from the SDS Setup File into the SDS plate document Note The SDS software uses the information from the SDS Setup File to automatically configure the plate grid and setup table with detector detector task marker and sample data For details on the SDS Setup File see page 26 IMPORTANT Modifying the contents of the SDS Setup File can corrupt the file making the file unusable that is you will not be able to access information for the TagMan Array card Look in la Custom Taqman Low Density Array x f amp e File name Sample TLDA Setup File txt Import Files of type Tab delimited Text
59. mfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NITE CHEMICAL HAZARD TaqMan Gene Expression Master Mix is harmful if swallowed Causes eye skin and respiratory tract irritation Read Material Safety Data Sheet and follow the handling instructions Wear protective eyewear clothing and gloves and use with adequate ventilation 1 For each sample determine the total number of reservoirs to be filled based on the format of your TaqMan Array card Notes 36 TaqMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Prepare the TagMan Array Micro Fluidic Card Per the table below calculate the total volume required for each reaction component volume for 1 fill reservoir x the total number of fill reservoirs per sample Include 12 5 excess volume in your calculations to compensate for the loss that occurs during pipetting Volume pL for Reaction Component 1 Fill Reservoir cDNA sample 30 to 1000 ng nuclease free water 50 0 TagMan Gene Expression Master Mix 50 0 or TagMan Universal PCR Master Mix with or without AmpErase UNG Total Volume 100 0 f Each sample specific PCR reaction mix should contain 30 to 1000 ng 0 3 to 10 ng uL of total RNA converted to cDNA The amount of cDNA to add depends upon the abundance of the specific gene transcript If frozen thaw th
60. n When you perform the experiment Chapter 4 the TagMan Gene Expression Assay used in the TagMan Array card may or may not detect genomic DNA The suffix of the assay ID indicates the assay placement and thus potential genomic DNA interference as described below Suffix Description m The assay s probe spans an exon junction The assay does not detect genomic DNA _s The assay s primers and probe are designed within a single exon The assay detects genomic DNA Note For s assays Applied Biosystems recommends that you take precautions to exclude genomic DNA from your RNA samples You can perform an RT minus experiment with the TaqMan Gene Expression Assay to test for genomic DNA contaminants g The assay may detect genomic DNA The assay s probe may span an exon junction or the assay s primers and probe may be within a single exon Note For g assays Applied Biosystems recommends that you take precautions to exclude genomic DNA from your RNA samples You can perform an RT minus experiment with the TaqMan Gene Expression Assay to test for genomic DNA contaminants mH The assay detects transcripts belonging to a gene family with high sequence homology The assay provides between 10 C and 15 C difference between the _sH target gene and the gene with the closest sequence homology Therefore the assay detects the target transcript with 1000 to 30 000 fold greater _gH discrimination sensitivity than the cl
61. n Array Micro Fluidic Cards User Guide iii Contents Appendix A Troubleshooting 00000 kk kk kk kk kk KK kK kk kk kk kk kk 53 Appendix B TagMan Endogenous Control Assays 57 Appendix C Required Materials aan 59 For Preparing Your Samples kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK kk 60 For Preparing the TagMan Array Micro Fluidic Card 0 a 61 For Running and Analyzing the TaqMan Array Micro Fluidic Card 62 For General Use nG 4 ana Pardee PP Gane Baek be ete ere AL 63 AppendixD Prevent Contamination 000 eee ee ees 65 Index DM CPP D 67 iv TagMan Array Micro Fluidic Cards User Guide Preface How to Use This Guide Purpose of This The Applied Biosystems TaqMan Array Micro Fluidic Cards User Guide provides Guide information for ordering TagMan Array Micro Fluidic Cards preparing and running the TaqMan Array cards on an Applied Biosystems 7900HT Fast Real Time PCR System then analyzing the results Audience This guide is intended for novice and experienced 7900HT Fast System users who perform quantitation experiments with the TaqMan Array cards using the comparative Cy AAC 7 method Text Conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For
62. nd stop position Inspect the affected wells after centrifuging and sealing the TaqMan Array card Note wells that contain bubbles and delete these wells from analysis TaqMan Array Micro Fluidic Cards User Guide 53 Appendix A Troubleshooting Observation Possible Cause Recommended Action After centrifuging Rare Reaction mix remains in a The fill port is blocked the fill Inspect the TaqMan Array card for a fill reservoir reservoir is defective blocked fill port or a pinched channel If the fill reservoir is defective contact Applied Biosystems Technical Support Filling is incomplete or not Centrifuge the TaqMan Array card again consistent for 1 minute If the reservoir filling is still not complete after the additional Excess reaction mix in a fill reservoir centrifuge cycle and you choose to process the TaqMan Array card further you should void the results for the affected fill reservoir sample aa IMPORTANT Do not exceed 1200 rpm or accumulated centrifugation times of more than 3 minutes Excessive centrifugation speeds and times may deform the TagMan Array card f Mm GR2162 Rare A fill reservoir is completely drained Some wells were not filled properly If you choose to process the TaqMan Array card further you should void the results for the affected fill reservoir Fill reservoir is completely drained sample
63. o www integromics com StatMiner php Notes TaqMan Array Micro Fluidic Cards User Guide 51 4 Chapter 4 Perform the Experiment Analyze the Experiment Notes 52 TaqMan Array Micro Fluidic Cards User Guide Appendix A Troubleshooting The following table lists some possible errors possible causes and recommended actions Observation Possible Cause Recommended Action After removing the TaqMan Array Micro Fluidic Card from its packaging Water condenses on the reaction The TaqMan Array card may not wells optical side of the TagMan have come to room temperature Array card before being removed from its packaging Remove condensation by lightly blowing on the reaction wells You can use room temperature pressurized nitrogen or an air blower IMPORTANT Be sure to remove all water condensation The exterior surface of the reaction wells optical side of the TagMan Array card must be free of water condensation After pipetting Too little PCR reaction mixture has The PCR mixture was not correctly gone into the fill reservoir pipetted into the fill reservoir Some of the PCR reaction mixture leaks out of the vent port in the fill reservoir Take care to correctly pipette the entire PCR reaction mixture 100 uL into the fill reservoir Add more sample Bubbles introduced into fill ports Air was introduced from pushing the pipette plunger in to its seco
64. oir Add master mix No amplification or poor amplification for a specific assay Low abundance gene Use more cDNA e Preamplify the cDNA The SDS v2 3 QC Flag is BPR Bad Passive Reference The Multicomponent Plot may show a gradual drop in ROX dye fluorescence Minor leaking from the wells due to inadequate sealing Use a steady even motion when using the staker Bubbles in well s Use proper pipetting techniques to avoid introducing bubbles when loading the fill reservoirs Note To determine if the data are usable Inspect the Multicomponent Plot The FAM and ROX dye signals should be parallel nspect the Amplification Plot There should not be any spikes or bumps in the exponential phase where the plot crosses the threshold Compare the C values to replicate wells If the values are comparable then the data are usable if the values are not comparable remove the well s as an outlier Notes TaqMan Array Micro Fluidic Cards User Guide 55 Appendix A Troubleshooting Observation Possible Cause Recommended Action Replicates have poor precision the standard deviation value is gt 0 5 for assays with a C value that is lt 30 Individual assay performance rising baseline or weak amplification Use the manual baseline setting Adjust the baseline and threshold settings then reanalyze Bubbles in well s Use proper pipett
65. ol assays for normalization PN 4331182 B TagMan Endogenous Control Assays A valid normalization or endogenous control is needed to correct for differences in RNA sampling and sample variation The ideal control is expressed consistently under Gene symbol Gene name Human assay ID Mouse assay ID Rat assay ID 18S Eukaryotic 18S rRNA Hs99999901 s1 Hs99999901 s1 Hs99999901 s1 ACTB Actin Beta cytoplasmic Hs99999903 m1 Mm00607939 s1 Rn00667896 m1 B2M Beta 2 microglobulin Hs99999907 m1 Mm00437762 m1 Rn00560865 m1 GAPDH Glyceradehyde 3 phosphate dehydrogenase Hs99999905 m1 Mm99999915 g1 Rn99999916 s1 GUSB Beta glucuronidase Hs99999908 m1 Mm00446953 m1 Rn00566655 m1 HMBS Hydromethylbilane synthase Hs00609297 m1 Mm00660262 g1 Rn00565886 m1 HPRT1 Hypoxanthine guanine phosphoribosyl transferase 1 Hs99999909 m1 Mm00446968 m1 Rn01527840 m1 IP08 Importin 8 Hs00183533_m1 Mm01255158_m1 Not Available PGK1 Phosphoglycerate kinase 1 Hs99999906_m1 Mm00435617_m1 Rn00821429_g1 POLR2A Polymerase RNA II DNA directed polypeptide A 220 kDa Hs00172187_m1 Mm00839493 m1 Rn01752026 m1 PPIA Peptidylprolyl isomerase A Hs99999904 m1 Mm02342430 g1 Rn00690933 m1 RPLPO Ribosomal protein large PO Hs99999902 m1 Mm00782638 s1 Rn01479927 g1 TBP TATA box
66. osest homologous transcript if both transcripts are present at the same copy number in a sample e The gH assay may detect genomic DNA e The mH assay does not detect genomic DNA e The sH assay detects genomic DNA u The assay s amplicon spans an exon junction and the probe sits completely in one of the spanned exons The assay does not detect genomic DNA ft The assay detects fusion transcripts that result from chromosomal translocation The probe and one primer are on one side of the fusion transcript breakpoint the second primer is on the other side of the fusion transcript breakpoint The assay does not detect genomic DNA at The assay is designed to detect a synthetic RNA transcript with a unique sequence that lacks homology to currently annotated biological sequences Notes TaqMan Array Micro Fluidic Cards User Guide 33 Chapter 3 Prepare Your Samples Store the cDNA Samples Perform Reverse To perform reverse transcription convert total RNA to cDNA Applied Biosystems Transcription recommends one of the kits listed below For information refer to the applicable kit protocol Kit Considerations Protocol High Capacity RNA to cDNA Kit Reaction components are premixed 2 tubes allowing for fewer pipetting steps 20X Enzyme Mix containing MuLV Reverse Transcriptase and RNase Inhibitor Protein 2X RT Buffer containing dNTP Mix oligo d T primers and random primers
67. ou suspect that the RNA contains RNase activity add RNase inhibitor to the reverse transcription reaction at a final concentration of 1 0 U UL It is not necessary to add RNase inhibitor to the reverse transcription reaction if the RNA was purified using the ABI PRISM 6100 Nucleic Acid PrepStation and Applied Biosystems nucleic acid purification reagents Nondenatured Note It is not necessary to denature the RNA Denaturation of the RNA my reduce the yield of cDNA for some gene targets To isolate total RNA from cellular samples Applied Biosystems recommends the TaqMan Gene Expression Cells to C Kit For procedures refer to www ambion com Note The Cells to C4 Kit includes the reverse transcription RNA to cDNA step If you use this kit it is not necessary to evaluate the RNA page 33 Ambion RNA Isolation Decision Tree For other recommendations on isolating total RNA refer to the Ambion RNA Isolation Decision Tree www ambion com techlib trees RNA index html 32 TagMan Array Micro Fluidic Cards User Guide Chapter 3 Prepare Your Samples Prepare Your Samples Evaluate the RNA Use high quality RNA that is free of contaminants for example proteins or detergents You can e Measure the UV absorbance A g o go to determine both quality and quantity Runan agarose gel to control the quality of 18S and 28S RNA Measure the RNA Integrity Number RIN Detection of genomic DNA During Amplificatio
68. overview TaqMan Array card 2 TaqMan Array Custom Micro Fluidic Card 2 TaqMan Gene Expression Assay 3 TaqMan Gene Sets 2 TaqMan Gene Signature Array 2 Q QUIKset control panel 40 R radioactive waste handling xii reaction mix prepare 36 troubleshooting 53 54 Real Time StatMiner Software 51 required materials 59 63 general use 63 running and analyzing 62 sample preparation 60 TaqMan Array card preparation 61 68 reverse transcription performing on samples 34 RNA convert to cDNA 34 detect genomic DNA 33 evaluate 33 isolate total 32 template guidelines 32 run the TagMan Array card 48 running multiple TagMan Array cards 46 S safety biological hazards xiii guidelines x xil samples detect genomic DNA 33 evaluate RNA 33 isolate total RNA 32 perform reverse transcription 34 prepare 32 34 RNA template guidelines 32 store 34 SDS Plate Document setup 46 SDS Setup File description 26 import 46 using 46 seal the TagMan Array card 42 StatMiner Software 51 store samples 34 T TaqMan Array card analyze the experiment 50 51 applications 4 centrifuge 39 diagram 4 downstream analysis 51 fill 38 filling guidelines 37 formats 22 25 instruments required to run 5 multiple cards 46 placing in bucket 39 product overview 2 reaction mix 36 recommended amounts of cDNA 36 review experiment results 50 run 48 seal 42 set up the SDS Plate Document 46 troubleshooting 53 56 TagMan Array Micro Fluidic Car
69. pecial handling and disposal limitations may apply TaqMan Array Micro Fluidic Cards User Guide Important Safety Information Biological Hazard Safety Biological Hazard Safety TAS BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available
70. plied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Chemical Safety Guidelines To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xi Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal TagMan Array Micro Fluidic Cards User Guide Important Safety Information About MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety inform
71. pter 2 Order the TagMan Array Micro Fluidic Card Order the TaqMan Array Micro Fluidic Card There are two ways to reorder the same TagMan Array Custom Micro Fluidic Cards or TaqMan Gene Sets Import a saved design from within the Configurator this page To use this method you must have previously saved a design step 11 on page 14 or added an order to your shopping basket step 12 on page 15 Note You can use this method if you have not yet submitted the order that is you added the order to your shopping basket but did not check out Reorder from your shopping basket page 21 To use this method you must have previously submitted an order step 12 on page 15 Import a saved design 1 Goto www appliedbiosystems com 2 In the I Want to Buy box click TaqMan Gene Expression Assays Plates amp Arrays I Want to Buy TagMan Gene Expression Assays Plates amp Arrays TaqMan MicroRNA Assays TaqMan SNP Genotyping Assays Real Time PCR Reagents amp Kits Custom Primers amp Probes Assays amp Custom Products unida 3 Under Arrays click 384 Well Arrays Micro Fluidic Card Gene Expression Assays amp Arrays Which gene expression assay is right for you Read our TaqMan Gene Expression Assays Product Guide PDF 1 0 MB Arrays TaqMan Custom 96 Well Arrays Plating Service Microplate lell Arrays Micro Fluidic Card Individual Assays Custom TaqMan
72. rcial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration or to sublicense repackage with other products or resell in any form is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS O 2009 2010 Life Technologies Corporation All rights reserved Trademarks of Life Technologies Corporation and its affiliated companies AB Design ABI PRISM Applied Biosystems Cells to CT FAM ROX AmpErase and TaqMan are registered trademarks of Roche Molecular Systems Inc Excel and Microsoft are registered trademarks of Microsoft Corporation StatMiner is a trademark of Integromics All other trademarks are the sole property of their respective owners Part Number 4400263 Rev C 07 2010 Chapter 1 Chapter 2 Chapter 3 Chapter 4 Contents PIBIdOS makaka KA KA ali xa aya wad Showa ee e d d d KANA V How to Use This Guide exe Ges sabes awk ated ND NBA n h le a RR E RD V How to Obtain More Information kk eee vi How to Obtain Support ehh vii Important Safety Information eee ix Safety Alert Words sesso
73. rols v A05 SMAD9 Hs00195441_m1 Gene Symbol Location AO6 sp1 Hs00293689 s1 18s rRNA A11 AO7 TGFBR2 Hs00559661 m1 il 10b update array Layout Reset Layout HERA BMP10 BMPR2 GDF10 SMAD1 SMAD9 SP1 TGFBR2 ACVR1 LEFTY2 TGFB1 185 SMAD3 B FST BMP3 ZFYVES SMADS PPP2CB MSTN ROCK1 GDF3 E2F5 GDF15 SKP1 E ACVRIB EP300 STUBi IL6 IL17F INHBB HIPK2 E2F4 INHBC SMAD2 BMP2 ACVR2A TGFBRAP1 RPS6KB2 MAPK3 TFDP1 TGFBR3 CULI GAPDH SMAD4 DCN B2M RHOA SMADE BMP10 BMPR2 GDF10 SMAD1 SMAD9 SP1 TGFBR2 ACVR1 LEFTY2 TGFB1 185 SMAD3 FST BMP3 ZFYVE9 SMADS PPP2CB MSTN ROCK1 GDF3 E2F5 GDF15 skpi o Gn 9 106 11 Optional Use one or more of the configuration tools Click Save This Design to save your current TagMan Array card design to the Applied Biosystems store workspace If your design is successfully saved this message appears at the top of the page You have successfully added your design to workspace Note For more information on the Applied Biosystems store workspace click the workspace link shown in the message Click Export Assay List then follow the prompts to export a list of your current assays to a simple text application txt file Click Print Array Layout then select File gt Print to print your current TaqMan Array card layout Notes 14 TagMan Array Micro Fluidic Cards User Guide Chapter
74. run as soon as possible After sealing there is no measurable well to well contamination for up to 64 hours Applied Biosystems recommends that you run replicate PCR reactions Refer to the TaqMan Gene Expression Master Mix Protocol Appendix B TaqMan Array Micro Fluidic Cards User Guide 37 Notes Chapter 4 Perform the Experiment Prepare the TaqMan Array Micro Fluidic Card Fill the TagMan Array card 1 Allow the TaqMan Array card to reach room temperature then carefully remove it from its packaging Note A minimum of 15 minutes at room temperature is required to equilibrate the card Place the TaqMan Array card on a lab bench with the foil side down Load 100 uL of the desired sample specific PCR reaction mix into a 100 uL micropipette Hold the micropipette in an angled position and place the tip in the fill port Note There is a fill port on the left arm of each fill reservoir it is the larger of the two holes Fill port Vent port UU IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port GR2158 Dispense the sample specific PCR reaction mix so that it sweeps in and around the fill reservoir toward the vent port IMPORTANT Pipette the entire 100 L L into the fill reservoir Be careful when pushing the micropipette plunger to its second stop position to expel the sample specific PCR reaction mix from the tip
75. says amp Custom Products Midas 3 Under Arrays click 384 Well Arrays Micro Fluidic Card Gene Expression Assays amp Arrays Which gene expression assay is right for you Read our TagMan Gene Expression Assays Product Guide PDF 1 0 MB Arrays TaqMan Custom 96 Well Arrays Plating Service Microplate 384 Well Arrays Micro Fluidic Card Individual Assays TaqMan Endogenous Controls TaqMan Gene Expression Assays 4 Click TaqMan Custom Array or TaqMan Gene Sets 384 Well Arrays Micro Fluidic Card TaqMan Custom TaqMan Gene Array Signature Arrays Human ABC Transporter TaqMan Gene Sets Array Human Alzheimer s Array and Mouse Alzheimer s Arraw 5 Optional On the product page click the Product Description tab for more information about the assays and the card layout Notes 10 TaqMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TagMan Array Micro Fluidic Card 2 Order the TaqMan Array Micro Fluidic Card 6 Click the Ordering Information tab then click Configure Ordering Information Product Description Literature Support TaqMan Gene Sets are genes grouped by path disease target class which can be ordei separately as a custom plate or configured directly onto TaqMan Arrays that enable you to perform hundreds of real time PCRr ions simultaneously on the Applied stems 7900HT Fast Real Time PCR n Simple to implement T
76. t click Connect to Instrument If the instrument tray is inside the instrument click Open Close to rotate the instrument tray to the OUT position Verify that the TagMan Array Micro Fluidic Card Thermal Cycling Block is installed in the instrument tray If the block is not installed you must Remove the existing block Install the TagMan Array Micro Fluidic Card Thermal Cycling Block Change the plate adapter For detailed information on changing the instrument blocks refer to the Applied Biosystems 7900HT Fast Real Time PCR System Maintenance and Troubleshooting Guide Place the prepared TaqMan Array card in the instrument tray with Well A1 at the top left corner of the tray and the notched corner at the top right The barcode toward the front of the instrument 48 TagMan Array Micro Fluidic Cards User Guide Chapter 4 Perform the Experiment 4 Run the TaqMan Array Micro Fluidic Card Well A1 Notched corner Barcode 8 Click Start Run The instrument tray rotates to the IN position During the run the instrument displays real time status information in the Instrument gt Real Time tabs and records the fluorescence emissions During the run you can view the data as the data are generated in real time 9 When the Run Complete dialog box appears click OK to close the dialog box click Open Close then remove the TaqMan Array card from the instrument tray Notes Taq
77. t contains the Array Information CD 3 Click then right click AIF ProdNum txt then select Open with Excel Notes TaqMan Array Micro Fluidic Cards User Guide 27 AIF columns Chapter 2 Order the TaqMan Array Micro Fluidic Card Array Information CD The table below describes the columns of the AIF Note In the table below N A appears in the Example column for fields that do not apply to TaqMan Array cards Field name Description of content Example Customer Name Your organization or institution University XYZ Sales Order Number A unique number that identifies the Applied Biosystems sales 1234567890 order Ship Date The date when the assay was packaged for shipment N A Delivery Number A unique bar code number that identifies the shipment N A Shipment ID i Note The shipment ID also appears in the plate ID Part Number A number that identifies the product line 4331182 Product Type The Applied Biosystems product line associated with the assay TaqMan Gene Expression Assays Assay ID An alphanumeric string that identifies the assay Hs00154374 m1 Lot Number A unique alphanumeric string that identifies the manufacturing A3579 batch to which the assay belongs Shipping Rack or The type of container in which the assay is shipped such as a 384 well 8 port Micro Plate Type 96 position or a 16 position tube rack Fluidic Card Shipping Rack or A
78. target assays 1 mandatory manufacturing control unique sample Replicates Port 1 m EJ 5 4 za B 3 m EI EI EI a als E a EB A 89 D ka a 5 6 m 145 146 147 Ca 149 BA T52 1 54 ES D E 10 161 Ha 163 764 los 5 T66 7 ma 145 147 uns ES 151 153 MM E A EHED IA 167 jos 185 186 18 q 190 BR Eg NEN 1 2 3 4 5 6 7 8 9 10 1 1 1 14 15 16 17 18 19 20 21 22 23 24 Format 384 380 target assays 4 mandatory manufacturing controls 1 unique sample Replicates Port EJ E 3 c 41 A E E T 59 eo 6 66 5 69 KM a EE 87 89 91 J 96 7 7 7 z mm EJ Em E E 3 Ez EN Notes TaqMan Array Micro Fluidic Cards User Guide 25 2 Array Information CD Chapter 2 Order the TaqMan Array Micro Fluidic Card Array Information CD SDS Setup File The Array Information CD is shipped with your TaqMan Array card It includes the following files SDS Setup File this page Array Map File page 27 Assay Information File AIF page 27 readme text file e PDF file of this guide the TaqMan Array Micro Fluidic Cards User Guide PDF file of the Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide The SDS Setup File is a text file txt included on the Array Information CD The SDS Setup File contains information specific to your TagMan Array card such as detector reporter and task information for each well When you set up the SDS plat
79. the card holder making sure that The fill reservoirs project upwards out of the card holder The reaction wells face the same direction as the This Side Out label Youuse blank balance cards to fill any open positions in the card holder Use the blank balance cards provided with the installation kits IMPORTANT Be sure to use the blank balance cards to fill any open positions The blank balance cards will balance the centrifuge and prevent damage to the card holder If the card holder is not completely filled the TaqMan Array card may become displaced during centrifugation resulting in uneven filling GR2155 Notes TaqMan Array Micro Fluidic Cards User Guide 39 4 Chapter 4 Perform the Experiment Prepare the TaqMan Array Micro Fluidic Card d Place a filled card holder in the bucket so that the This Side Out label faces the front of the bucket Sorvall emblem here 2 Set the centrifuge a Power on the centrifuge b Use the front panel controls to set the bucket type to 15679 IMPORTANT To ensure that the maximum rotational speed stays within the manufacturer s specified limits be sure to set the correct bucket type c Use the front panel controls to set the following operations parameters Parameter EASYSet touchpad Baa d Up ramp rate 9 3 Down ramp rate 9 N A Rotational speed 1200 rpm 331 x g 1200 rpm Centrifugation time 2 x 1 m
80. ts Format 48 47 target assays 1 mandatory manufacturing control 8 unique samples Replicates Port 1 lt a 2 l lt 2 3 lt 3 4 3 4 5 B 6 6 7 ar 8 i TB 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Format 64 63 target assays 1 mandatory manufacturing control 2 unique samples Replicates Port 1 2 1 3 4 5 6 2 N 5 55 0g P 1 2 3 4 5 6 Y 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Format 96a 95 target assays 1 mandatory manufacturing control 4 unique samples Replicates Port AKI 1 E D E 2 1E i T 2 K 6 t ra 8 Ilo 8 P Notes 24 TagMan Array Micro Fluidic Cards User Guide Chapter 2 Order the TaqMan Array Micro Fluidic Card 2 Available Formats Format 96b 95 target assays 1 mandatory manufacturing control 2 unique samples Replicates Port 3 3 E Ad E 23 E z 3 34 3 38 Hn m KI 1 24 2 Gr 34 3 43 464 Bae 3 53 gt 5 o o 63 67 6 AN E 3 2 lt 4 a LB gt J IG 2 M 7 TIN 1 o 8 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Format 192 191
81. uidic Cards User Guide 35 4 Chapter 4 Perform the Experiment Prepare the TaqMan Array Micro Fluidic Card Prepare the TaqMan Array Micro Fluidic Card Prepare the PCR reaction Fill the TaqMan Array Centrifuge the TaqMan gt Seal the TaqMan Array mix CDNA master mix card with the reaction mix Array card page 39 card page 42 this page page 37 Note When preparing the TaqMan Array card see the product part numbers and sizes under For Preparing the TagMan Array Micro Fluidic Card on page 61 Recommended For the amplification PCR reactions Applied Biosystems recommends that you use Amounts of cDNA 30to 1000 ng 0 3 to 10 ng L of cDNA converted from total RNA per fill reservoir The amount to use depends on the expression level of your target genes and the number of target copies per well that you need to detect For example you can use 1000 ng 10 ng uL per fill reservoir to detect genes with low expression However the cDNA concentration will be high 10 ng uL be sure to use high quality cDNA without inhibitors 100 to 200 ng per fill reservoir to detect genes with moderate expression 30 to 50 ng per fill reservoir to detect genes with moderate to high expression Thesame amount of cDNA sample for all reactions Prepare the PCR CHEMICAL HAZARD TagMan Universal PCR Master Mix may Reaction Mix cause eye and skin irritation Exposure may cause disco
82. xt x Cancel 5 Save the SDS plate document a Select File Save As b Navigate to a save location c Enter a name for the SDS plate document d For Files of Type select SDS 7900HT Document sds or SDS 7900HT Template Document sdt Note You can save the plate document as an SDS plate document sds or SDS template sdt Saving the plate document as an SDS template is recommended when you want to create duplicate plate documents for a series of TaqMan Array cards with identical assay configurations For more information on SDS templates refer to the SDS Online Help e Click Save Notes TaqMan Array Micro Fluidic Cards User Guide 47 4 Chapter 4 Perform the Experiment Run the TaqMan Array Micro Fluidic Card Perform the Run Notes A TagMan Array Micro Fluidic Card Thermal Cycling Block must be installed on the instrument For more information see Instrument Overview on page 5 AN i weh Surface may be hot Open the SDS plate document in the SDS software Select the Instrument tab Use the default thermal cycling conditions as shown in the Thermal Cycler tab Note When you selected 384 Well TagMan Low Density Array step 3 on page 46 the SDS software automatically set the appropriate thermal cycling conditions for the TaqMan Array cards Select the Real Time tab then verify that is displayed in the status bar If the software is not connected to the instrumen

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TaqMan&reg; Array Micro Fluidic Cards

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TaqMan® Array Micro Fluidic Cards