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Chapter 7

Prepare the Comparative C

Reactions

Prepare the Reaction Plate

Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve

and Comparative C

Experiments

146

Notes

For More

Information

For more information on preparing the reaction mix, refer to the protocol appropriate for
the reagents you are using in the PCR reactions:

• TaqMan

Gene Expression Assays Protocol

• Custom TaqMan

Gene Expression Assays Protocol

Prepare the Reaction Plate

Prepare the reactions for each replicate group, then transfer them to the reaction plate.
Use the plate layout displayed in the 7500 software.

About the

Example

Experiment

For the comparative C

example experiment:

• A MicroAmp

Optical 96-Well Reaction Plate is used.

• The reaction volume is 50

L/well.

• The reaction plate contains:

– 96 Unknown wells

– 0 Negative Control wells

– 0 Empty wells

• The plate layout that is automatically generated by the 7500 software is used:

Required

Materials

• Microcentrifuge tubes

• Pipettors

• Pipette tips

• Reaction mixes (from page 145)

• Water

• Samples (from page 143)

• MicroAmp

Optical 96-Well Reaction Plate

• MicroAmp

™

Optical Adhesive Film

• Centrifuge

Summary of Contents for 7500

Page 1: ...Applied Biosystems 7500 7500 Fast Real Time PCR System Relative Standard Curve and Comparative CT Experiments Getting Started Guide ...

Page 2: ......

Page 3: ...lative Standard Curve Experiment Prepare the Relative Standard Curve Reactions Run the Relative Standard Curve Experiment Analyze the Relative Standard Curve Experiment Design the Comparative CT Experiment Prepare the Comparative CT Reactions Run the Comparative CT Experiment Analyze the Comparative CT Experiment Getting Started Guide ...

Page 4: ...conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS Applera Applied Biosystems AB Design MicroAmp Primer Ex...

Page 5: ...ical Hazard Safety xxi Workstation Safety xxi Safety and Electromagnetic Compatibility EMC Standards xxii Chapter 1 Get Started 1 About the 7500 7500 Fast System 2 Supported Consumables 4 About Relative Standard Curve and Comparative CT Experiments 7 How to Use This Guide 12 About the Example Experiments 14 Example Experiment Workflow 17 Chapter 2 Design the Relative Standard Curve Experiment 19 C...

Page 6: ... Prepare for the Run 67 Enable the Notification Settings Optional 69 Start the Run 71 Monitor the Run 72 Unload the Instrument 74 Chapter 5 Analyze the Relative Standard Curve Experiment 75 Chapter Overview 76 Section 5 1 Review Results 77 Analyze the Experiment 78 View the Standard Curve 82 View the Amplification Plot 85 View the Gene Expression Plot and Well Table 93 Publish the Data 97 Section ...

Page 7: ...e the Reaction Mix 144 Prepare the Reaction Plate 146 Chapter 8 Run the Comparative CT Experiment 151 Chapter Overview 152 Prepare for the Run 153 Run the Experiment 154 Chapter 9 Analyze the Comparative CT Experiment 155 Chapter Overview 156 Section 9 1 Review Results 157 Analyze the Experiment 158 View the Gene Expression Plot and Well Table 162 View the Amplification Plot 166 Publish the Data 1...

Page 8: ... Started Guide for Relative Standard Curve and Comparative CT Experiments vi Appendix A Alternate Experiment Workflows 203 Advanced Setup Workflow 204 QuickStart Workflow 206 Template Workflow 208 Export Import Workflow 210 Bibliography 213 Glossary 215 Index 229 ...

Page 9: ...urve and Comparative CT Experiments 4387783 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments 4387779 Applied Biosystems 7500 7500 Fast Real Time PCR System Maintenance Guide Explains how to install and maintain the 7500 7500 Fast system Intended for laboratory staff responsible for the installation and maintenance of the 7500 7500 Fast sys...

Page 10: ... For example Select File Open User Attention Words Two user attention words appear in Applied Biosystems user documentation Each word 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 r...

Page 11: ...ence Card 4374204 Performing a Custom TaqMan SNP Genotyping Assay for 96 Well Plates Quick Reference Card 4371394 Performing a TaqMan Drug Metabolism Genotyping Assay for 96 Well Plates Quick Reference Card 4367636 Pre Developed TaqMan Assay Reagents Allelic Discrimination Protocol 4312214 TaqMan Drug Metabolism Genotyping Assays Protocol 4362038 TaqMan SNP Genotyping Assays Protocol 4332856 Docum...

Page 12: ...c Assays Protocol Submission Guidelines 4367671 Custom TaqMan SNP Genotyping Assays Protocol 4334431 Power SYBR Green PCR Master Mix and RT PCR Protocol 4367218 Pre Developed TaqMan Assay Reagents Allelic Discrimination Protocol 4312214 Primer Express Software Version 3 0 Getting Started Guide 4362460 SYBR Green PCR and RT PCR Reagents Protocol 4304965 SYBR Green PCR Master Mix and RT PCR Reagents...

Page 13: ...tion go to http www appliedbiosystems com then click the link for Support See How to Obtain Support on page xi How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order App...

Page 14: ...jury 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 Except for IMPORTANTs each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applie...

Page 15: ...s is not a protected ground terminal Indicates the Off position of the main power switch Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates ...

Page 16: ...ts chimiques dangeureux Lire les fiches techniques de sûreté de matériels avant la manipulation des produits CAUTION Hazardous waste Refer to MSDS s and local regulations for handling and disposal ATTENTION Déchets dangereux Lire les fiches techniques de sûreté de matériels et la régulation locale associées à la manipulation et l élimination des déchets WARNING Hot lamp AVERTISSEMENT Lampe brûlant...

Page 17: ... System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Locations of Warnings The Applied Biosystems 7500 7500 Fast Real Time PCR System contains warnings at the locations shown below Attention Physical hazard Physical hazard Attention Attention Attention ...

Page 18: ... or more people Things to consider before lifting the computer and or the monitor Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with y...

Page 19: ...nt and waste bottles 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 information you need to store handle transport and dispose of the chemicals safely Each time you r...

Page 20: ...act 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 c...

Page 21: ...ntainer 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 Min...

Page 22: ...ower supply in your laboratory FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument Power ELECTRICAL HAZARD Grounding circuit continuity is vital for the safe operation of equipment Never operate equipment with the grounding conductor disconnected ELECTRICAL HAZARD Use properly configured and approved line c...

Page 23: ... 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 guidel...

Page 24: ...plies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators European Safety and EMC Standards Safety This instrument meets European requirements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General ...

Page 25: ... Supported Consumables 4 About Relative Standard Curve and Comparative CT Experiments 7 How to Use This Guide 12 About the Example Experiments 14 Example Experiment Workflow 17 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500...

Page 26: ...lluminates all wells of the reaction plate within the instrument exciting the fluorophores in each reaction 2 Emission The instrument optics collect the residual fluorescence emitted from the wells of the reaction plate The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths 3 Collection The instrument assembles a digital representat...

Page 27: ...n the Help select Help 7500 Software Help in the 7500 software Genotyping experiments Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Genotyping Experiments Presence absence experiments Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Presence Absence Experiments Standard curve experiments Refer to the Appli...

Page 28: ...e Strip 0 2 mL MicroAmp Optical 8 Cap Strip 4316567 4323032 MicroAmp Optical Tube without Cap 0 2 mL MicroAmp Reaction Tube with Cap 0 2 mL N8010933 N8010540 Microamp Splash Free Support Base N8010531 MicroAmp Adhesive Film Applicator MicroAmp Cap Installing Tool Handle MicroAmp Multi Removal Tool 4333183 4330015 4313950 Consumable Consumable A MicroAmp Optical 96 Well Reaction Plate 0 2 mL E Micr...

Page 29: ... standard reagents 7 Consumable Part Number MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL MicroAmp Optical Adhesive Film 4346906 4311971 MicroAmp Fast 8 Tube Strip 0 1 mL MicroAmp Optical 8 Cap Strip 4358293 4323032 Microamp Splash Free Support Base N8010531 MicroAmp Adhesive Film Applicator MicroAmp Cap Installing Tool Handle MicroAmp Multi Removal Tool 4333183 4330015 4313950 ...

Page 30: ... 0 2 mL PN N2070540 MicroAmp Optical 8 Tube Strip 0 2 mL PN 4316567 also called standard reaction tube strips IMPORTANT Fast reaction plates and tubes do not fit into the standard sample block correctly and will result in loss of data 7500 Fast system MicroAmp Fast Optical 96 Well Reaction Plate PN 4346906 also called Fast reaction plates MicroAmp Fast 8 Tube Strip 0 1 mL PN 4358293 also called Fa...

Page 31: ...et quantity in the samples and in the reference sample The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample Relative standard curve experiments are commonly used to Compare expression levels of a gene in different tissues Compare expression levels of a gene in a treated sample vs an untreated s...

Page 32: ...y used to Compare expression levels of a gene in different tissues Compare expression levels of a gene in a treated sample vs an untreated sample Compare expression levels of wild type alleles with those of mutated alleles Components The following components are required when setting up PCR reactions for comparative CT experiments Sample The sample in which the quantity of the target is unknown Re...

Page 33: ...e change in expression of a target in a sample relative to the same target in a reference sample Best for assays that have suboptimal PCR efficiency Advantage Relative levels of target in samples can be determined without the use of a standard curve if the PCR efficiencies of the target and endogenous control are relatively equivalent Reduced reagent usage More space available in the reaction plat...

Page 34: ...ANT SYBR Green reagents cannot be used for multiplex PCR 1 vs 2 Step RT PCR You can perform reverse transcription RT and PCR in a single reaction 1 step or in separate reactions 2 step The reagent configuration you use depends on whether you are performing 1 or 2 step RT PCR In 1 step RT PCR RT and PCR take place in one buffer system which provides the convenience of a single tube preparation for ...

Page 35: ...e stranded DNA binding dye to detect PCR products as they accumulate during PCR cycles Advantages Economical no probe needed Allows for melt curve analysis to measure the Tm of all PCR products Can be used for either 1 or 2 step RT PCR Limitations Binds nonspecifically to all double stranded DNA sequences To avoid false positive signals check for nonspecific product formation using melt curve or g...

Page 36: ...for performing your own experiments Using This Guide as a Tutorial Using the example experiment data provided with the 7500 software you can use this guide as a tutorial for performing a relative standard curve or comparative CT experiment on a 7500 7500 Fast system Follow the procedures in the appropriate chapters shown in the table below For more information see About the Example Experiments on ...

Page 37: ...rkflows Workflow Description See Design Wizard Set up a new experiment with guidance from the 7500 software The Design Wizard guides you through best practices as you create your own experiment The Design Wizard is recommended for new users Note Design options are more limited in the Design Wizard than in Advanced Setup Chapter 2 or Chapter 6 Advanced Setup Set up a new experiment using advanced o...

Page 38: ...ndard curve is set up for c myc target The standard used for the standard dilution series is a cDNA sample of known quantity prepared from RNA isolated from lung tissue One standard curve is set up for GAPDH endogenous control The standard used for the standard dilution series is a cDNA sample of known quantity prepared from RNA isolated from lung tissue The experiment is designed for singleplex P...

Page 39: ...periment is designed for singleplex PCR where the targets EGR3 MAOB OGDH OSGEP and SERPING1 and endogenous control 18S assays are performed in separate wells Reactions are set up for 2 step RT PCR The High Capacity cDNA Reverse Transcription Kit is used for reverse transcription the TaqMan Universal PCR Master Mix is used for PCR Primer probe sets are selected from the Applied Biosystems TaqMan Ge...

Page 40: ...le experiment file that contains run data and in a comparative CT example study file that contains the data from multiple comparative CT experiments The data files install with the 7500 software and are on your computer at drive Applied Biosystems software name experiments Comparative Ct Example eds drive Applied Biosystems software name experiments Comparative Ct Study Example edm where drive is ...

Page 41: ...eries 4 Prepare the reaction mix for each target assay 5 Prepare the reaction plate Run the Experiment Chapter 4 1 Prepare for the run 2 Enable the notification settings Optional 3 Start the run 4 Monitor the run 5 Unload the instrument Start Relative Standard Curve Experiment Design the Experiment Chapter 6 1 Create a new experiment 2 Define the experiment properties 3 Define the methods and mate...

Page 42: ...shold 2 View the quality summary 3 Omit wells 4 View the Multicomponent Plot 5 View the Raw Data Plot Comparative CT CT Experiment continued from page 17 Analyze the Experiment Chapter 9 Section 1 Review Results 1 Analyze 2 View the Gene Expression Plot well table 3 View the Amplification Plot 4 Publish the data Section 2 Troubleshoot If Needed 1 View the analysis settings adjust the baseline thre...

Page 43: ...Define the Methods and Materials 24 Set Up the Targets 26 Set Up the Standards 29 Set Up the Samples 31 Set Up the Relative Quantitation Settings 34 Set Up the Run Method 35 Review the Reaction Setup 36 Order Materials for the Experiment 43 Finish the Design Wizard 46 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fa...

Page 44: ... the example experiment Note When you design your own experiments you can select alternate workflows see Using This Guide with Your Own Experiments on page 13 Example Experiment Workflow Design the Experiment Chapter 2 1 Create a new experiment 2 Define the experiment properties 3 Define the methods and materials 4 Set up the targets 5 Set up the standards 6 Set up the samples 7 Set up the relativ...

Page 45: ... and Comparative CT Experiments Notes Create a New Experiment Create a new experiment using the Design Wizard in the 7500 software Create an Experiment 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of the 7500 software 2 In the Home screen click Design Wizard to open the Design Wizard 2 ...

Page 46: ...riment The experiment is identified as an example The instrument that is selected to run the experiment is the 7500 instrument A MicroAmp Optical 96 Well Reaction Plate is used The experiment type is quantitation Complete the Experiment Properties Screen 1 Click the Experiment Name field then enter Relative Standard Curve Example Note The experiment header is updated with the experiment name that ...

Page 47: ...s in the Barcode field Optional Enter a user name to identify the owner of the experiment You can enter up to 100 characters in the User Name field Optional Enter comments to describe the experiment You can enter up to 1000 characters in the Comments field Select the instrument you are using to run the experiment 7500 96 Wells 7500 Fast 96 Wells Note You can use 7500 software v2 0 to design experi...

Page 48: ...tandard curve quantitation method is used TaqMan reagents are used The standard ramp speed is used in the instrument run cDNA prepared from total RNA isolated from liver and kidney tissues is the template type You must first perform reverse transcription to convert the RNA to cDNA see Prepare the Template on page 51 Complete the Methods and Materials Screen 1 In the Methods and Materials screen se...

Page 49: ...e target The SYBR Green dye fluoresces when it binds to double stranded DNA SYBR Green dye is often part of the SYBR Green master mix added to the reaction If you use SYBR Green dye select the Include Melt Curve check box to perform melt curve analysis of the amplified target IMPORTANT Although you can use other fluorescence based reagents on the 7500 7500 Fast system you must design your experime...

Page 50: ...get About the Example Experiment In the relative standard curve example experiment Two targets are quantified in the reaction plate The Set Up Standards check box is selected When this check box is selected the software automatically displays the Standards screen after you complete the Targets screen In the Standards screen you can set up a standard curve for each target assay see Set Up the Stand...

Page 51: ... FAM default c In the Quencher drop down list select NFQ MGB default d In the Color field leave the default 4 Set up the Target 2 experiment a Click the Enter Target Name cell then enter GAPDH b In the Reporter drop down list select FAM default c In the Quencher drop down list select NFQ MGB default d In the Color field leave the default 5 Click Next Note For all targets leave blank the Optional E...

Page 52: ...or variable sample mass nucleic acid extraction efficiency reverse transcription efficiency and pipette calibration errors Note that Each sample type for example each tissue in a study comparing multiple tissues requires an endogenous control If samples are on multiple plates each reaction plate must have an endogenous control Additionally every plate must include an endogenous control for every s...

Page 53: ...of known quantity prepared from RNA isolated from lung tissue For each standard curve Five points are used in the standard curve Three replicates are used for each point Replicates are identical reactions containing identical reaction components and volumes The starting quantity is 200 ng and the serial factor is 1 10 Complete the Standards Screen 1 In the Standards screen click the How many point...

Page 54: ...d quantities affects the amplification efficiency calculations carefully consider the appropriate range of standard quantities for your assay For more accurate measurements of amplification efficiency use a broad range of standard quantities such as between 105 and 106 If you specify a broad range of quantities for the standards you need to use a PCR product or a highly concentrated template such ...

Page 55: ... The samples contain unknown quantities of the c myc gene target and GAPDH gene endogenous control Three replicates are used The replicates are identical reactions containing identical reaction components and volumes Three negative controls are used The negative control reactions contain water instead of sample and should not amplify Complete the Samples Screen 1 In the Samples screen click the Ho...

Page 56: ...lative standard curve experiment Identify each sample with a unique name and color You can enter up to 100 characters in the Sample Name field Enter the number of replicates identical reactions to set up Applied Biosystems recommends three replicates for each sample reaction Enter the number of negative control reactions to set up Applied Biosystems recommends three negative control reactions for ...

Page 57: ...ore targets per well design your experiment using Advanced Setup instead of the Design Wizard Set Up Biological Replicate Groups 1 Select Specify Biological Replicate Groups 2 Enter the number of biological replicate groups that you want to test in the reaction plate 3 For each biological replicate group click the cell in the Biological Group Name column then enter a name for the biological group ...

Page 58: ... the Which target do you want to use as the endogenous control drop down list 3 Click Next Design Guidelines When you design your own relative standard curve experiment Select a reference sample from your previously created samples Set Up the Samples on page 31 Amplification results from the samples are compared to the amplification results from the reference sample to determine relative expressio...

Page 59: ...you can edit the default run method or replace it with one from the Run Method library About the Example Experiment In the relative standard curve example experiment the default run method is used without edits Review the Run Method Screen 1 In the Run Method screen select either the Graphical View tab default or Tabular View tab 2 Make sure the Reaction Volume Per Well field displays 50 µL 3 Make...

Page 60: ...or More Information For more information on The Run Method library or on completing the Run Method screen Open the 7500 Software Help by clicking or pressing F1 Using Advanced Setup See Advanced Setup Workflow on page 204 Review the Reaction Setup In the Reaction Setup screen select the assay type if using TaqMan reagents then review the calculated volumes for preparing the PCR reactions standard ...

Page 61: ... default 2 In the Select Target pane select c myc 3 In the Assay Type drop down list select Inventoried Made to Order 4 Make sure the Reaction Volume Per Well field displays 50 µL 5 Make sure the Excess Reaction Volume field displays 10 6 In the Reactions for c myc pane a Make sure the Master Mix Concentration field displays 2 0 b Make sure the Assay Mix Concentration field displays 20 0 c Review ...

Page 62: ...Reaction Setup screen select the Reaction Mix Calculations tab default 2 In the Select Target pane select GAPDH 3 In the Assay Type drop down list select Inventoried Made to Order 4 Make sure the Reaction Volume Per Well field displays 50 µL 5 Make sure the Excess Reaction Volume field displays 10 6 In the Reactions for GAPDH pane a Make sure the Master Mix Concentration field displays 2 0 b Make ...

Page 63: ... drop down list select ng per µL default c Review the calculated volumes for preparing the standard dilution series Component Volume µL for 1 Reaction Master Mix 2 0 25 0 Assay Mix 20 0 2 50 Sample 10 or Standard 5 0 The sample or standard volume is limited to 10 of the total reaction volume H2O 17 50 Total Volume 50 0 1 2 3 5 4 6b 6a 6c Dilution Point Source Source Volume µL Diluent Volume µL Tot...

Page 64: ...lculations tab 1 In the Reaction Setup screen select the Sample Dilution Calculations tab 2 Click the Diluted Sample Concentration 10 for Reaction Mix field then enter 50 3 In the unit drop down list select ng µL default 4 Review the calculated volumes for the sample dilutions 7b 7a 7c Sample Name Stock Concentration ng µL Sample Volume µL Diluent Volume µL Total Volume of Diluted Sample µL Liver ...

Page 65: ...rative CT Experiments Notes You can print detailed reaction setup instructions then save the instructions for Chapter 3 Prepare the Relative Standard Curve Reactions To print Reaction Setup instructions 1 Click Print Reaction Setup 2 In the Print Reaction Setup Instructions dialog box select Detailed Reaction Setup Instructions Include Plate Layout Use sample color 3 Click Print 4 Click Next 1 2 3...

Page 66: ...ntrations For SYBR Green reagents edit the master mix forward primer and reverse primer concentrations For 1 step RT PCR edit the reverse transcriptase concentration Review the reaction mix components for each target If you are running Fast PCR reactions make sure you use Fast master mix in the PCR reactions If you are running standard PCR reactions make sure you use standard master mix in the PCR...

Page 67: ...The 7500 software recommends the materials to order based on your experiment design It is assumed that you will design your experiment order your materials then prepare Chapter 3 and run Chapter 4 the reaction plate when your materials arrive About the Example Experiment In the relative standard curve example experiment the recommended materials are MicroAmp Optical 96 Well Reaction Plate MicroAmp...

Page 68: ...e Applied Biosystems Store 3 Optional Click Print Materials List to send the materials list to your printer 4 Optional Create a shopping list a Select the check box next to each of the following items MicroAmp Optical 96 Well Reaction Plate MicroAmp Optical Adhesive Film MicroAmp Splash Free Support Base TaqMan Universal PCR Master Mix 2 No AmpErase UNG Hs00153408_m1 c myc Assay Mix Hs99999905_m1 ...

Page 69: ...red materials and that the quantities are correct then click Order Materials in List b In the Order Materials Log In dialog box enter your user name and password for the Applied Biosystems Store then click Log In and Submit Note If you do not have an account with the Applied Biosystems Store click Register Now to create an account c When you are connected to the Applied Biosystems Store follow the...

Page 70: ...ript are turned on For More Information For more information on completing the Materials List screen open the 7500 Software Help by clicking or pressing F1 Finish the Design Wizard Finish the Design Wizard review the plate layout then select an exit option About the Example Experiment The 7500 software automatically selects locations for the wells in the reaction plate In the relative standard cur...

Page 71: ...d wells 6 Negative control wells 48 Empty wells Note If the plate layout is incorrect click Return to the Wizard and check your entered values 3 Click Save Experiment 4 In the Save Experiment dialog box enter Relative Standard Curve Example Setup eds in the File name drop down list then click Save The example experiment is saved and closed and you are returned to the Home screen IMPORTANT Do not s...

Page 72: ...t dialog box Default save location Select Tools Preferences then select the Defaults tab In the Data Folder field browse to then select the desired location For More Information For more information on using Advanced Setup see Advanced Setup Workflow on page 204 Click To Save Experiment Save and close the experiment without making any further changes or starting the run Start Run for This Experime...

Page 73: ...overs Chapter Overview 50 Prepare the Template 51 Prepare the Sample Dilutions 53 Prepare the Standard Dilution Series 55 Prepare the Reaction Mix 58 Prepare the Reaction Plate 60 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7...

Page 74: ... to prepare the PCR reactions for the relative standard curve example experiment Example Experiment Workflow Design the Experiment Chapter 2 Start Relative Standard Curve Experiment Run the Experiment Chapter 4 Analyze the Experiment Chapter 5 End Experiment Prepare the Reactions Chapter 3 1 Prepare the template 2 Prepare the sample dilutions 3 Prepare the standard dilution series 4 Prepare the re...

Page 75: ...ty cDNA Reverse Transcription Kit Required Materials One of the following Ambion starter packs for RNA isolation Kit Contents Ambion Catalog Number qRT PCR Starter Pack RNAlater Tissue Collection RNA Stabilization Solution AM7020 RNaseZap Wipes AM9786 RT PCR Grade Water nuclease free AM9935 Silencer Validated siRNA Std Purity AM51331 One of the following RNA sample preparation products RNAqueous 4...

Page 76: ...ystems High Capacity cDNA Reverse Transcription Kits Protocol to 1 Prepare the RT master mix CHEMICAL HAZARD 10 RT Buffer may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Prepare the cDNA reactions 3 Perform reverse transcription on a thermal cycler High Capacity cDNA Kit RNAlater Tissue...

Page 77: ...ing RNA or gDNA templates Refer to the protocol for the purification reagents that you select To locate Applied Biosystems purification reagents visit http www appliedbiosystems com Prepare the Sample Dilutions Perform sample dilutions before adding the samples to the final reaction mix Dilute the samples using the volumes that were calculated by the 7500 software To complete the Sample Dilution C...

Page 78: ... 5 Place the diluted samples on ice until you prepare the reaction plate Preparation Guidelines When you prepare your own relative standard curve experiment Sample dilutions may be necessary because the sample volume is limited to 10 of the total reaction volume in the 7500 software You must perform the sample dilutions before adding the samples to the final reaction mix For optimal performance of...

Page 79: ...he relative standard curve example experiment The standard stock concentration is 100 ng µL The volumes calculated in the software for both the c myc and GAPDH assays are Required Materials Water to dilute the standards Microcentrifuge tubes Pipettors Pipette tips Standard stock Vortexer Centrifuge Prepare the Standard Dilution Series for the c myc Assay 1 Label a separate microcentrifuge tube for...

Page 80: ...iefly 5 In the c myc Std 3 tube a Using a new pipette tip add 2 02 µL of dilution 2 to the c myc Std 3 tube b Vortex Std 3 for 3 to 5 seconds then centrifuge the tube briefly 6 In the c myc Std 4 tube a Using a new pipette tip add 2 02 µL of dilution 3 to the c myc Std 4 tube b Vortex Std 4 for 3 to 5 seconds then centrifuge the tube briefly 7 In the c myc Std 5 tube a Using a new pipette tip add ...

Page 81: ...e b Vortex Std 3 for 3 to 5 seconds then centrifuge the tube briefly 6 In the GAPDH Std 4 tube a Using a new pipette tip add 2 02 µL of dilution 3 to the GAPDH Std 4 tube b Vortex Std 4 for 3 to 5 seconds then centrifuge the tube briefly 7 In the GAPDH Std 5 tube a Using a new pipette tip add 2 02 µL of dilution 4 to the GAPDH Std 5 tube b Vortex Std 5 for 3 to 5 seconds then centrifuge the tube b...

Page 82: ...mponents for the PCR reactions However when you prepare the reaction mix in this section include only the master mix assay mix and water Add the sample or standard when you prepare the reaction plate see Prepare the Reaction Plate on page 60 About the Example Experiment For the relative standard curve example experiment The reaction mix components are TaqMan Universal PCR Master Mix 2 c myc Assay ...

Page 83: ... Mix GAPDH Reaction Mix 2 For the c myc assay add the required volumes of each component to the c myc Reaction Mix tube 3 For the GAPDH assay add the required volumes of each component to the GAPDH Reaction Mix tube 4 Mix the reaction mix in each tube by gently pipetting up and down then cap each tube 5 Centrifuge the tubes briefly to remove air bubbles 6 Place the reaction mixes on ice until you ...

Page 84: ...robes Prior to use Mix the master mix thoroughly by swirling the bottle Resuspend the assay mix by vortexing then centrifuge the tube briefly Thaw any frozen samples by placing them on ice When the samples are thawed resuspend the samples by vortexing then centrifuge the tubes briefly For More Information For more information on preparing the reaction mix refer to the protocol appropriate for the ...

Page 85: ... page 56 Samples from page 54 MicroAmp Optical 96 Well Reaction Plate MicroAmp Optical Adhesive Film Centrifuge Prepare the Reaction Plate 1 For each target prepare the negative control reactions a To an appropriately sized tube add the volumes of reaction mix and water listed below b Mix the reaction by gently pipetting up and down then cap the tube c Centrifuge the tube briefly to remove air bub...

Page 86: ...0 µL of the unknown sample reaction to the appropriate wells in the reaction plate 4 Seal the reaction plate with optical adhesive film Tube Standard Reaction Reaction Mix Reaction Mix Volume µL Standard Standard Volume µL 1 c myc Std 1 c myc reaction mix 148 5 c myc Std 1 16 5 2 c myc Std 2 c myc reaction mix 148 5 c myc Std 2 16 5 3 c myc Std 3 c myc reaction mix 148 5 c myc Std 3 16 5 4 c myc S...

Page 87: ...ment Make sure you use the appropriate consumables Make sure the arrangement of the PCR reactions matches the plate layout displayed in the 7500 software You can either Accept the plate layout automatically generated by the software or Use Advanced Setup to change the plate layout in the software If you use optical adhesive film seal each reaction plate as follows a Place the reaction plate onto t...

Page 88: ...e end of the end tab and pull up and away sharply Repeat for the other end tab h Repeat step f to ensure a tight evaporation free seal While applying firm pressure run the edge of the applicator along all four sides of the outside border of the film Note Optical adhesive films do not adhere on contact The films require the application of pressure to ensure a tight seal i Inspect the reaction plate...

Page 89: ...s chapter covers Chapter Overview 66 Prepare for the Run 67 Enable the Notification Settings Optional 69 Start the Run 71 Monitor the Run 72 Unload the Instrument 74 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software H...

Page 90: ...s chapter explains how to perform a run on the Applied Biosystems 7500 7500 Fast Real Time PCR System Example Experiment Workflow Design the Experiment Chapter 2 Start Relative Standard Curve Experiment Prepare the Experiment Chapter 3 Analyze the Experiment Chapter 5 End Experiment Run the Experiment Chapter 4 1 Prepare for the run 2 Optional Enable the notification settings 3 Start the run 4 Mon...

Page 91: ... in Chapter 2 then loading the sealed reaction plate into the 7500 7500 Fast instrument Open the Example Experiment 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of the 7500 software 2 In the Home screen click Open 3 In the Open dialog box navigate to the experiments folder default at drive Applie...

Page 92: ...e that the plate is properly aligned in the holder 3 Place the reactions in the precision plate holder PPH If you use A reaction plate Place the reaction plate in the PPH with well A1 at the back left corner Reaction tube strips Place the tube strips in the PPH for tube strips Note Fast Reaction 8 tube strips can be used only on the 7500 Fast system For the 7500 system use MicroAmp Optical 8 Tube ...

Page 93: ...em Leave columns 2 3 10 and 11 empty 4 Close the tray door Apply pressure to the right side of the tray and at an angle Enable the Notification Settings Optional Enable the notification settings so that the 7500 software alerts you by email when the 7500 7500 Fast instrument begins and completes a run or if an error occurs during a run Enabling the notifications settings is optional and does not a...

Page 94: ...ification Settings 3 Select Yes for Enable Notifications 4 Select the events that will generate notifications a Select Instrument Error b Select Run Completed 5 In the Enter e mail addresses for notifications field enter scientist mycompany com supervisor mycompany com technician mycompany com 6 In the Outgoing Mail Server SMTP field enter smtp mycompany com 7 Set the authentication settings a Sel...

Page 95: ...when the instrument completes a run Obtain e mail addresses of those whom you want to be notified IMPORTANT Separate addresses with a comma Contact your systems administrator or information technology department if you need The e mail addresses of users who will receive notifications A network address for a simple mail transfer protocol SMTP server on the local area network LAN A user name and pas...

Page 96: ...ays normalized dye fluorescence Rn as a function of cycle number The figure below shows the Amplification Plot screen as it appears during the example experiment To view data in the Amplification Plot screen select the wells that you want to view in the View Plate Layout tab To Action Stop the run 1 In the 7500 software click STOP RUN 2 In the Stop Run dialog box click one of the following Stop Im...

Page 97: ...ted cycle determined from previous similar experiments that were run using the same reagents under the same conditions If you notice abnormal amplification or a complete absence of florescence troubleshoot the error as explained in the 7500 Software Help click or press F1 About the Run Method Screen This screen displays the method for the run in progress The software updates the Run Status field t...

Page 98: ...e Help click or press F1 Unload the Instrument When your 7500 7500 Fast instrument displays the Run Complete unload the reaction plate from the instrument Unload the Reaction Plate PHYSICAL INJURY HAZARD During operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 Push the tray door to open it 2...

Page 99: ...2 View the Amplification Plot 85 View the Gene Expression Plot and Well Table 93 Publish the Data 97 Section 5 2 Troubleshoot If Needed 99 View the Analysis Settings 100 View the QC Summary 103 Omit Wells from the Analysis 105 View the Multicomponent Plot 107 View the Raw Data Plot 109 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosy...

Page 100: ...ive standard curve example experiment includes three flagged wells The flags indicate common problems that you may encounter when performing your own experiments Procedures are provided for viewing the flags and omitting one of the wells Example Experiment Workflow Design the Experiment Chapter 2 Start Relative Standard Curve Experiment Prepare the Reactions Chapter 3 Run the Experiment Chapter 4 ...

Page 101: ...00 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Notes Section 5 1 Review Results This section covers Analyze the Experiment 78 View the Standard Curve 82 View the Amplification Plot 85 View the Gene Expression Plot and Well Table 93 Publish the Data 97 ...

Page 102: ...le for the example experiment is on your computer at drive Applied Biosystems software name experiments Relative Standard Curve Example eds where drive is the computer hard drive on which the 7500 software is installed software name is the current version of the 7500 software Analyze the Example Experiment 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software s...

Page 103: ...g within the analysis screens Preparing the Experiment for Review The example file contains a single outlier well A9 that has been omitted from the analysis for you To use the experiment file with this chapter you must include the well and reanalyze the experiment 1 Select the View Plate Layout tab then select the outlier well that is marked well A9 2 Right click well A9 then select Include 3 Clic...

Page 104: ...een on your computer To reanalyze the data select all the wells in the plate layout then click Analyze Navigation Tips How to Select Wells To display specific wells in the analysis screens select the wells in the View Plate Layout tab 1 To select wells of a specific type in the Select Wells With drop down list select Sample Target or Task then select the sample target or task name 2 To select a si...

Page 105: ...ative CT Experiments Notes How to Display Multiple Plots 1 In the navigation pane select Analysis Multiple Plots View 2 To display four plots click Show plots in a 2 2 matrix 3 To display two plots in rows click Show plots in two rows 4 To display two plots in columns click Show plots in two columns 5 To display a specific plot select the plot in the drop down list above each plot display 4 2 3 5 ...

Page 106: ...fficient CT values View the Standard Curve 1 In the navigation pane select Analysis Standard Curve Note If no data are displayed click Analyze 2 Display all 96 wells in the Standard Curve screen by clicking the upper left corner of the plate layout in the View Plate Layout tab 3 In the Plot Settings tab select All in the Target drop down list default 4 In the Plot Settings tab select Default in th...

Page 107: ...e CT Experiments Notes 7 Check that all samples are within the standard curve In the example experiment all samples blue dots are within the standard curve red dots 8 Check the CT values a Select the View Well Table tab b In the Group By drop down list select Replicate c Look at the values in the CT column In the example experiment the CT values are within the expected range 8 and 35 6 3 4 5 7 8b ...

Page 108: ...ors PCR inhibitors in the reaction can reduce amplification efficiency R2 values correlation coefficient A measure of the closeness of fit between the regression line and the individual CT data points of the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points An R2 value 0 99 is desirable CT values The PCR cycle number at which the fluorescenc...

Page 109: ...y and examine irregular amplification and to view threshold and baseline values for the run Rn vs Cycle Rn is the fluorescence from the reporter dye normalized to the fluorescence from the passive reference This plot displays Rn as a function of cycle number You can use this plot to identify and examine irregular amplification CT vs Well CT is the PCR cycle number at which the fluorescence meets t...

Page 110: ...ote If no data are displayed click Analyze 2 Display the GAPDH wells in the Amplification Plot screen a Select the View Plate Layout tab b In the Select Wells With drop down lists select Target then GAPDH 3 In the Plot Settings tab of the Amplification Plot a In the Plot Type drop down list select Rn vs Cycle default b In the Plot Color drop down list select Well c Click Show a legend for the plot...

Page 111: ...ive CT Experiments Notes 4 View the baseline values Plot Settings a In the Graph Type drop down list select Linear b Select the Baseline check box to show the start cycle and end cycle c Verify that the baseline is set correctly The end cycle should be set a few cycles before the cycle number where significant fluorescence is detected In the example experiment the baseline is set correctly 4b 3c 3...

Page 112: ...tandard Curve and Comparative CT Experiments 88 Notes 5 View the threshold values a In Plot Settings tab select Log in the Graph Type drop down list b Select GAPDH in the Target drop down list c Select the Threshold check box to show the threshold default d Verify that the threshold is set correctly In the example experiment the threshold is in the exponential phase 5c 5a 5b ...

Page 113: ...T Experiments Notes 6 Locate any outliers a In the Plot Type drop down list select CT vs Well b Look for outliers in the amplification plot In the example experiment there are no outliers for GAPDH 7 Repeat steps 2 through 6 for the c myc target In the example experiment there is one outlier for c myc well A9 You omit this well in the troubleshooting section Omit Wells from the Analysis on page 10...

Page 114: ...t a typical amplification plot A typical amplification plot has four distinct sections a Plateau phase b Linear phase c Exponential geometric phase d Baseline IMPORTANT Experimental error such as contamination or pipetting errors can produce atypical amplification curves that can result in incorrect baseline and threshold value calculations by the 7500 software Therefore Applied Biosystems recomme...

Page 115: ...ncrease the standard deviation of the replicate groups Threshold Set Too Low The threshold is set below the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar up into the exponential phase of the curve Threshold Set Too High The threshold is set above the exponential phase of t...

Page 116: ...ally adjust the baseline and or threshold see View the Analysis Settings on page 100 For More Information For more information on the Amplification Plot screen open the 7500 Software Help by clicking or pressing F1 Baseline Examples Baseline Set Correctly The amplification curve begins after the maximum baseline Baseline Set Too Low The amplification curve begins too far to the right of the maximu...

Page 117: ... the gene expression profile Two plots are available RQ vs Target Groups the relative quantitation RQ values by target Each sample is plotted for each target You can display the plot on a linear log10 Ln or log2 scale RQ vs Sample Groups the relative quantitation RQ values by sample Each target is plotted for each sample You can display the plot on a linear log10 Ln or log2 scale The Well Table di...

Page 118: ...on pane select Analysis Gene Expression Note If no data are displayed click Analyze 2 In the Gene Expression Plot screen a In the Plot Type drop down list select RQ vs Sample b In the Graph Type drop down list select Log10 c In the Orientation drop down list select Vertical Bars 3 Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide ...

Page 119: ...metic average of the technical replicate CT values Normalized Qty Mean The point estimate of the normalized quantities computed at the replicate level as the geometric mean Normalized Qty Std Err The confidence interval based variability associated with the normalized quantities computed at the replicate level as the geometric standard error of the mean RQ The relative level of gene expression for...

Page 120: ...ample experiment there is one outlier well A9 You will omit this well in the troubleshooting section Omit Wells from the Analysis on page 105 Note To show hide columns in the Well Table select deselect the column name in the Show in Table drop down list Analysis Guidelines When you analyze your own relative standard curve experiment look for Differences in gene expression as a fold change relative...

Page 121: ...nd Comparative CT Experiments Notes Publish the Data You can publish the experiment data in several ways Save the plot as an image file Print the plot Print the reaction plate layout Create slides Print a report Export data to applications such as Microsoft Excel and Microsoft PowerPoint For information on performing these tasks open the 7500 Software Help by clicking or pressing F1 ...

Page 122: ...5 Analyze the Relative Standard Curve Experiment Publish the Data Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments 98 Notes ...

Page 123: ...7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Notes Section 5 2 Troubleshoot If Needed This section covers View the Analysis Settings 100 View the QC Summary 103 Omit Wells from the Analysis 105 View the Multicomponent Plot 107 View the Raw Data Plot 109 ...

Page 124: ... default analysis settings in the 7500 software are not suitable for your experiment you can change the settings then reanalyze your experiment About the Example Experiment In the standard curve example experiment the default analysis settings are used without changes View the Settings 1 In the navigation pane select Analysis 2 Click Analysis Settings to open the Analysis Settings dialog box 3 In ...

Page 125: ...on page 101 Reject outliers Select the algorithm to use to determine RQ Min Max values confidence level or standard deviations Flag Settings Use this tab to Adjust the sensitivity so that more wells or fewer wells are flagged Change the flags that are applied by the 7500 software Advanced Settings Use this tab to change baseline settings well by well Set Multiple Endogenous Controls You can select...

Page 126: ...for Relative Standard Curve and Comparative CT Experiments 102 Notes 3 In the Select Multiple Endogenous Controls dialog box select the targets that you want to use as endogenous controls then click OK For More Information For more information on the analysis settings open the 7500 Software Help by pressing F1 when the Analysis Settings dialog box is open 1 2 3 ...

Page 127: ... flags generated by the experiment data In the example experiment the HIGHSD flags has been generated for wells A7 A8 and A9 View the QC Summary 1 In the navigation pane select Analysis QC Summary Note If no data are displayed click Analyze 2 Review the Flags Summary In the example experiment three wells are flagged 3 In the Flag Details table look in the Frequency and Wells columns to determine w...

Page 128: ...shooting link to view information on correcting the flag You can change the flag settings Adjust the sensitivity so that more wells or fewer wells are flagged Change the flags that are applied by the 7500 software For More Information For more information on the QC Summary screen or on flag settings open the 7500 Software Help by clicking or pressing F1 Flag Description AMPNC Amplification in nega...

Page 129: ...rs from the analysis About the Example Experiment In the example experiment you use the Well Table to remove well A9 from the analysis The CT of well A9 deviates significantly from those of the related technical replicates which generated the HIGHSD flag due to the degree of variation in their CT values see View the QC Summary on page 103 Omit Wells 1 In the navigation pane select Analysis Amplifi...

Page 130: ...uidelines When you analyze your own relative standard curve experiment carefully view the replicate groups for outliers If needed remove outliers manually using the Well Table See Omit Wells on page 105 to remove the outliers in your experiment For More Information For more information on omitting wells from the analysis open the 7500 Software Help by clicking or pressing F1 Within the Help search...

Page 131: ...egative control wells View the Plot 1 In the navigation pane select Analysis Multicomponent Plot Note If no data are displayed click Analyze 2 Display the unknown and standard wells one at a time in the Multicomponent Plot screen a Select the View Plate Layout tab b Select one well in the plate layout the well is shown in the Multicomponent Plot screen Note If you select multiple wells the Multico...

Page 132: ...tive Standard Curve and Comparative CT Experiments 108 Notes 6 View the ROX dye signal In the example experiment the ROX dye signal is constant throughout the PCR process which indicates typical data 7 Select the negative control wells one at a time and check for amplification In the example experiment no amplification occurs in the negative control wells 2b 3 2a 4 5 6 7 ...

Page 133: ...on For more information on the Multicomponent Plot screen open the 7500 Software Help by clicking or pressing F1 View the Raw Data Plot The Raw Data Plot screen displays the raw fluorescence not normalized for each optical filter for the selected wells during each cycle of the real time PCR About the Example Experiment In the relative standard curve example experiment you review the Raw Data Plot ...

Page 134: ...le increase occurs in the signal from filter 1 which corresponds to the FAM dye The filters are Analysis Guidelines When you analyze your own relative standard curve experiment look for the following in each filter Characteristic signal growth No abrupt changes or dips For More Information For more information on the Raw Data Plot screen open the 7500 Software Help by clicking or pressing F1 3 4 F...

Page 135: ... 114 Define the Methods and Materials 116 Set Up the Targets 119 Set Up the Samples 121 Set Up the Relative Quantitation Settings 124 Set Up the Run Method 125 Review the Reaction Setup 127 Order Materials for the Experiment 131 Finish the Design Wizard 134 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Tim...

Page 136: ...ters for the example experiment Note When you design your own experiments you can select alternate workflows see Using This Guide with Your Own Experiments on page 13 Example Experiment Workflow Design the Experiment Chapter 6 1 Create a new experiment 2 Define the experiment properties 3 Define the methods and materials 4 Set up the targets 5 Set up the samples 6 Set up the relative quantitation ...

Page 137: ... Comparative CT Experiments Notes Create a New Experiment Create a new experiment using the Design Wizard in the 7500 software Create an Experiment 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of the 7500 software 2 In the Home screen click Design Wizard to open the Design Wizard 2 ...

Page 138: ...CT example experiment The experiment is identified as an example The 7500 instrument is selected to run the experiment A MicroAmp Optical 96 Well Reaction Plate is used The experiment type is quantitation Complete the Experiment Properties Screen 1 Click the Experiment Name field then enter Comparative CT Example Note The experiment header is updated with the experiment name you entered 2 Leave th...

Page 139: ...Enter a user name to identify the owner of the experiment You can enter up to 100 characters in the User Name field Optional Enter comments to describe the experiment You can enter up to 1000 characters in the Comments field Select the instrument you are using to run the experiment 7500 96 Wells 7500 Fast 96 Wells Note You can use 7500 software v2 0 to design experiments for the 7500 7500 Fast ins...

Page 140: ...e CT quantitation method is used TaqMan reagents are used The standard ramp speed is used in the instrument run cDNA prepared from total RNA isolated from liver kidney brain and universal tissues is the template type You must first perform reverse transcription to convert the RNA to cDNA see Prepare the Template on page 139 Complete the Methods and Materials Screen 1 Select Comparative CT CT as th...

Page 141: ...he target The TaqMan probe is designed to hybridize to the target and fluoresce when the target is amplified Select SYBR Green Reagents if you want to use SYBR Green reagents to detect amplification and quantify the amount of target in the samples SYBR Green reagents consist of two primers and SYBR Green dye The primers are designed to amplify the target The SYBR Green dye fluoresces when it binds...

Page 142: ...ompleting the Methods and Materials screen Open the 7500 Software Help by clicking or pressing F1 Determining PCR efficiencies Open the 7500 Software Help by clicking or pressing F1 Within the Help search as follows a Select the Search tab b Enter PCR efficiency c Click List Topics d Double click Determine Amplification Efficiency Using Advanced Setup See Advanced Setup Workflow on page 204 Using ...

Page 143: ...e experiment the targets are EGR3 MAOB OGDH OSGEP and SERPING1 Complete the Targets Screen 1 Click the How many targets do you want to quantify in the reaction plate field then enter 6 Note The number of rows in the target assays table is updated with the number you entered 2 Set up the Target 1 assay a Click the Enter Target Name cell then enter 18S b In the Reporter drop down list select FAM def...

Page 144: ... sample type for example each tissue in a study comparing multiple tissues requires an endogenous control If samples are on multiple plates each plate must have an endogenous control Additionally every reaction plate must include an endogenous control for every sample type on the reaction plate You can select multiple endogenous controls for a single plate See Set Multiple Endogenous Controls on p...

Page 145: ... and volumes No negative controls are used Negative control reactions contain water instead of sample and should not amplify The software automatically includes three negative controls for each target assay IMPORTANT Although the example experiment does not use negative controls Applied Biosystems recommends them Complete the Samples Screen 1 Click the How many samples do you want to test in the r...

Page 146: ...You can enter up to 100 characters in the Sample Name field Enter the number of replicates identical reactions to set up Applied Biosystems recommends three replicates for each sample reaction Set up biological replicate groups see Set Up Biological Replicate Groups on page 123 Biological replicates allow you to assess the representative nature of your results as they relate to the population bein...

Page 147: ...ological replicate groups when analyzing comparative CT experiments as part of a study see Define Replicates on page 192 1 Select Specify Biological Replicate Groups 2 Enter the number of biological replicate groups that you want to test in the reaction plate 3 For each biological replicate group click the cell in the Biological Group Name column then enter a name for the biological group For exam...

Page 148: ...use as the endogenous control drop down list select 18S 3 Click Next Design Guidelines When you design your own comparative CT experiment Select a reference sample from your previously created samples Set Up the Samples on page 121 Amplification results from the samples are compared to the amplification results from the reference sample to determine relative expression Select an endogenous control...

Page 149: ...If needed you can edit the default run method or replace it with one from the Run Method library About the Example Experiment In the comparative CT example experiment the default run method is used without edits Review the Run Method Screen 1 Select either the Graphical View tab default or Tabular View tab 2 Make sure the Reaction Volume Per Well field displays 50 µL 3 Make sure the thermal profil...

Page 150: ...upports reaction volumes from 10 to 30 µL Review the thermal profile Make sure the thermal profile is appropriate for your reagents If you are performing 1 step RT PCR include a reverse transcription step If your experiment requires a different thermal profile edit the thermal profile or replace the run method with one from the Run Method library The Run Method library is included in the 7500 soft...

Page 151: ...per well is 50 µL The excess reaction volume is 10 The reaction components are TaqMan Universal PCR Master Mix 2 Assay Mix 20 for each target 18S EGR3 MAOB OGDH OSGEP and SERPING1 Sample Water The diluted sample concentration is 50 ng µL The sample stock concentration is 100 ng µL Complete the Reaction Setup Screen IMPORTANT Perform the following steps for each target assay in the reaction plate C...

Page 152: ... the remaining targets listed in the Selected Targets pane EGR3 MAOB OGDH OSGEP and SERPING1 Complete the Sample Dilution Calculations Tab 1 Select the Sample Dilution Calculations tab 2 Click the Diluted Sample Concentration 10 for Reaction Mix field then enter 50 0 3 In the unit drop down list select ng µL default Component Volume µL for 1 Reaction Master Mix 2 0 25 00 Assay Mix 20 0 2 50 Sample...

Page 153: ...ns for use in Chapter 7 Prepare the Comparative CT Reactions 1 Click Print Reaction Setup 2 In the Print Reaction Setup Instructions dialog box select Detailed Reaction Setup Instructions Include Plate Layout Use sample color 3 Click Print to print the reaction setup instructions 4 Click Next Sample Name Stock Concentration ng µL Sample Volume µL Diluent Volume µL Total Volume of Diluted Sample µL...

Page 154: ...ystems recommends an excess reaction volume of at least 10 Review the reaction mix concentrations for each target If needed For TaqMan reagents edit the master mix and assay mix concentrations For SYBR Green reagents edit the master mix forward primer and reverse primer concentrations For 1 step RT PCR edit the reverse transcriptase concentration Review the reaction mix components for each target ...

Page 155: ...t order your materials then prepare Chapter 7 and run Chapter 8 the reaction plate when your materials arrive About the Example Experiment In the comparative CT example experiment the recommended materials are MicroAmp Optical 96 Well Reaction Plate MicroAmp Optical Adhesive Film MicroAmp Splash Free Support Base TaqMan Universal PCR Master Mix 2 No AmpErase UNG 18S Assay Mix Hs99999901_s1 EGR3 As...

Page 156: ...Create a shopping list a Select the check box next to each of the following items MicroAmp Optical 96 Well Reaction Plate MicroAmp Optical Adhesive Film MicroAmp Splash Free Support Base TaqMan Universal PCR Master Mix 2 No AmpErase UNG 18S Assay Mix Hs99999901_s1 EGR3 Assay Mix Hs00231780_m1 MAOB Assay Mix Hs00168533_m1 OGDH Assay Mix Mm00803121_m1 OSGEP Assay Mix Hs00215099_m1 SERPING1 Assay Mix...

Page 157: ...rials and that the quantities are correct then click Order Materials in List b In the Order Materials Log In dialog box enter your user name and password for the Applied Biosystems Store then click Log In and Submit Note If you do not have an account with the Applied Biosystems Store click Register Now to create an account c When you are connected to the Applied Biosystems Store follow the prompts...

Page 158: ...der IMPORTANT Make sure that cookies and JavaScript are turned on For More Information For more information on completing the Materials List screen open the 7500 Software Help by clicking or pressing F1 Finish the Design Wizard To finish the Design Wizard review the plate layout then select an exit option About the Example Experiment The 7500 software automatically selects locations for the wells ...

Page 159: ... closed Note For the example experiment do not perform the run at this time Finish the Design Wizard 1 At the bottom of the 7500 software screen click Finish Designing Experiment 2 In the Review Plate Layout for Experiment window review the plate layout Make sure there are 96 Unknown wells 0 Negative Control wells 0 Empty wells Note If the plate layout is incorrect click Return to the Wizard then ...

Page 160: ...t the appropriate exit option By default experiments are saved to drive Applied Biosystems software name experiments To change the Save location for a specific experiment Navigate to the desired location using the Save Experiment dialog box Default save location Select Tools Preferences then select the Defaults tab In the Data Folder field browse to then select the desired location For More Inform...

Page 161: ...his chapter covers Chapter Overview 138 Prepare the Template 139 Prepare the Sample Dilutions 142 Prepare the Reaction Mix 144 Prepare the Reaction Plate 146 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help ...

Page 162: ...for the comparative CT CT example experiment and it provides guidelines for how to prepare the PCR reactions for your own comparative CT experiment Example Experiment Workflow Design the Experiment Chapter 6 Start Comparative CT CT Experiment Run the Experiment Chapter 8 Analyze the Experiment Chapter 9 End Experiment Prepare the Reactions Chapter 7 1 Prepare the template 2 Prepare the sample dilu...

Page 163: ... cDNA reverse transcribed from total RNA samples using the High Capacity cDNA Reverse Transcription Kit Required Materials One of the following Ambion starter packs for RNA isolation Kit Contents Catalog Number qRT PCR Starter Pack RNAlater Tissue Collection RNA Stabilization Solution AM7020 RNaseZap Wipes AM9786 RT PCR Grade Water nuclease free AM9935 Silencer Validated siRNA Std Purity AM51331 C...

Page 164: ...tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Prepare the cDNA reactions 3 Perform reverse transcription on a thermal cycler PCR Starter Pack RT PCR Grade Water nuclease free AM9935 DNAZap AM9890 Choice of RNA sample preparation products as listed above under the qRT PCR Starter Pack See above High Capacity cDNA Kit RN...

Page 165: ... High Capacity cDNA Reverse Transcription Kit RNA Purified total RNA or mRNA extracted from tissue or sample Genomic DNA gDNA Purified gDNA already extracted from tissue or sample For More Information For more information on Preparing cDNA templates Refer to the Applied Biosystems High Capacity cDNA Reverse Transcription Kits Protocol The protocol is not shipped with the High Capacity cDNA Reverse...

Page 166: ...the total reaction volume in the 7500 software Because the total reaction volume is 50 µL reaction the sample volume is 5 µL reaction The stock sample concentration is 100 ng µL After diluting the sample according to the Sample Dilutions Calculations table the sample has a concentration of 50 ng µL Adding 5 µL at this concentration to the final reaction mix volume of 50 µL yields a 1 concentration...

Page 167: ...n Guidelines When you prepare your own comparative CT experiment Sample dilutions may be necessary because the sample volume is limited to 10 of the total reaction volume in the 7500 software You must perform the sample dilutions before adding the samples to the final reaction mix For optimal performance of TaqMan Gene Expression Assays or Custom TaqMan Gene Expression Assays use 10 to 100 ng of c...

Page 168: ... in this section include only the master mix assay mix and water Add the sample when you prepare the reaction plate see Prepare the Reaction Plate on page 146 About the Example Experiment For the comparative CT example experiment The reaction mix components are TaqMan Universal PCR Master Mix 2 18S Assay Mix 20 EGR3 Assay Mix 20 MAOB Assay Mix 20 OGDH Assay Mix 20 OSGEP Assay Mix 20 SERPING1 Assay...

Page 169: ...elines When you prepare your own comparative CT experiment If your experiment includes more than one target assay prepare the reaction mix for each target assay separately Include excess volume in your calculations to provide excess volume for the loss that occurs during reagent transfers Applied Biosystems recommends an excess volume of at least 10 Include all required components Prepare the reag...

Page 170: ... Reaction Plate Prepare the reactions for each replicate group then transfer them to the reaction plate Use the plate layout displayed in the 7500 software About the Example Experiment For the comparative CT example experiment A MicroAmp Optical 96 Well Reaction Plate is used The reaction volume is 50 µL well The reaction plate contains 96 Unknown wells 0 Negative Control wells 0 Empty wells The p...

Page 171: ...niversal 22 0 5 EGR3 Brain EGR3 Reaction Mix 198 0 Liver 22 0 6 EGR3 Kidney EGR3 Reaction Mix 198 0 Kidney 22 0 7 EGR3 Liver EGR3 Reaction Mix 198 0 Brain 22 0 8 EGR3 Universal EGR3 Reaction Mix 198 0 Universal 22 0 9 MAOB Brain MAOB Reaction Mix 198 0 Liver 22 0 10 MAOB Kidney MAOB Reaction Mix 198 0 Kidney 22 0 11 MAOB Liver MAOB Reaction Mix 198 0 Brain 22 0 12 MAOB Universal MAOB Reaction Mix ...

Page 172: ...parative CT experiment Make sure you use the appropriate consumables Make sure the arrangement of the PCR reactions matches the plate layout displayed in the 7500 software You can either Accept the plate layout automatically generated by the software or Use Advanced Setup to change the plate layout in the software If you use optical adhesive film seal each reaction plate as follows a Place the rea...

Page 173: ...d of the end tab and pull up and away sharply Repeat for the other end tab h Repeat step f to ensure a tight evaporation free seal While applying firm pressure run the edge of the applicator along all four sides of the outside border of the film Note Optical adhesive films do not adhere on contact The films require the application of pressure to ensure a tight seal i Inspect the reaction plate to ...

Page 174: ... Prepare the Comparative CT Reactions Prepare the Reaction Plate Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments 150 Notes ...

Page 175: ...un the Comparative CT Experiment This chapter covers Chapter Overview 152 Prepare for the Run 153 Run the Experiment 154 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help ...

Page 176: ... chapter explains how to perform a run on the Applied Biosystems 7500 7500 Fast Real Time PCR Systems Example Experiment Workflow Design the Experiment Chapter 6 Start Comparative CT CT Experiment Prepare the Experiment Chapter 7 Analyze the Experiment Chapter 9 End Experiment Run the Experiment Chapter 8 1 Prepare for the run 2 Enable the notification settings Optional 3 Start the run 4 Monitor t...

Page 177: ...apter 6 then loading the sealed reaction plate into the 7500 7500 Fast instrument Open the Example Experiment 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of the 7500 software 2 In the Home screen click Open 3 In the Open dialog box navigate to the experiments folder default at drive Applied Bios...

Page 178: ...ive Standard Curve and Comparative CT Experiments 154 Notes Run the Experiment Perform the following procedures in Chapter 4 for the comparative CT experiment Load the Reaction Plate into the Instrument on page 68 Enable the Notification Settings Optional on page 69 Start the Run on page 71 Monitor the Run on page 72 Unload the Instrument on page 74 ...

Page 179: ...ish the Data 174 Section 9 2 Troubleshoot If Needed 175 View the Analysis Settings 176 View the QC Summary 179 Omit Wells from the Analysis 181 View the Multicomponent Plot 182 View the Raw Data Plot 185 Section 9 3 Perform a Study of Multiple Experiments 187 Section Overview 188 Design a Study 189 Analyze the Study 195 Publish the Data 202 Note For more information about any of the topics discuss...

Page 180: ... how to analyze multiple experiments as a study Example Experiment Workflow Analyze the Experiment Chapter 9 Section 1 Review Results 1 Analyze 2 View the Gene Expression Plot well table 3 View the Amplification Plot 4 Publish the data Section 2 Troubleshoot If Needed 1 View the analysis settings adjust the baseline threshold 2 View the quality summary 3 Omit wells 4 View the Multicomponent Plot 5...

Page 181: ... 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Notes Section 9 1 Review Results This section covers Analyze the Experiment 158 View the Gene Expression Plot and Well Table 162 View the Amplification Plot 166 Publish the Data 174 ...

Page 182: ...d on a 7500 7500 Fast instrument The data file for the example experiment is on your computer at drive Applied Biosystems software name experiments Comparative Ct Example eds where drive is the computer hard drive on which the 7500 software is installed software name is the current version of the 7500 software Analyze the Example Experiment 1 Double click 7500 software or select Start All Programs...

Page 183: ...he data using the default analysis settings 6 See Navigation Tips on page 160 for information on navigating within the analysis screens Guidelines When you analyze your own comparative CT experiment Immediately after a run the 7500 software automatically analyzes the data using the default analysis settings then displays the Amplification Plot screen on your computer To reanalyze the data select a...

Page 184: ...nalysis screens select the wells in the View Plate Layout tab as follows 1 To select wells of a specific type use the Select Wells With drop down lists select Sample Target or Task then select the sample target or task name 2 To select a single well click the well in the plate layout 3 To select multiple wells click drag over the desired wells or CTRL click or Shift click the desired wells in the ...

Page 185: ...he Multiple Plots view to display up to four plots simultaneously To navigate within the Multiple Plots view 1 In the navigation pane select Analysis Multiple Plots View 2 To display four plots click Show plots in a 2 5 2 matrix 3 To display two plots in rows click Show plots in two rows 4 To display two plots in columns click Show plots in two columns 5 To display a specific plot select the plot ...

Page 186: ...an display the plot on a linear log10 Ln or log2 scale RQ vs Sample Groups the relative quantitation RQ values by sample Each target is plotted for each sample You can display the plot on a linear log10 Ln or log2 scale The Well Table displays data for each well in the reaction plate including The sample name target name task and dyes The calculated threshold cycle CT normalized fluorescence Rn an...

Page 187: ...In the Graph Type drop down list select Log10 c In the Orientation drop down list select Vertical Bars 3 Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend In the example experiment the expression of EGR3 MAOB OGDH OSGEP and SERPING1 in the liver kidney and universal samples are displayed relative to the expression ...

Page 188: ...he arithmetic average of the technical replicate CT values CT Mean The arithmetic average of the technical replicate CT values for the sample replicate group CT SD The sample standard deviation of the sample replicate group level CT values CT The calculated CT value for the replicate group associated with the test sample RQ The calculated relative level of gene expression for the replicate group a...

Page 189: ...ate the precision of the replicate groups In the example experiment there are no outliers Note To show hide columns in the Well Table select deselect the column name in the Show in Table drop down list Analysis Guidelines When you analyze your own comparative CT experiment look for Differences in gene expression as a fold change relative to the reference sample Standard deviation in the replicate ...

Page 190: ...y and examine irregular amplification and to view threshold and baseline values for the run Rn vs Cycle Rn is the fluorescence from the reporter dye normalized to the fluorescence from the passive reference This plot displays Rn as a function of cycle number You can use this plot to identify and examine irregular amplification CT vs Well CT is the PCR cycle number at which the fluorescence meets t...

Page 191: ...If no data are displayed click Analyze 2 Display the 18S wells in the Amplification Plot screen a Select the View Plate Layout tab b In the Select Wells With drop down lists select Target then 18S 3 In the Amplification Plot screen Plot Settings a In the Plot Type drop down list select Rn vs Cycle default b In the Plot Color drop down list select Well default c Click Show a legend for the plot Not...

Page 192: ...aseline Start check box to show the start cycle and end cycle c Verify that the baseline is set correctly The end cycle should be set a few cycles before the cycle number where significant florescence is detected In the example experiment the baseline is set correctly Note By default the Amplification Plot displays the data for all targets present in the experiment Consequently the plot overlays t...

Page 193: ...nts Notes 5 View the threshold values a In Plot Settings tab select Log in the Graph Type drop down list b Select 18S in the Target drop down list c In the Options tab select the Threshold check box to show the threshold d Verify that the threshold is set correctly The example experiment contains multiple thresholds one for each target assay The thresholds for all targets occur in the exponential ...

Page 194: ...d Comparative CT Experiments 170 Notes 6 Locate any outliers a In Plot Settings tab select CT vs Well in the Plot Type drop down list b Look for outliers in the amplification plot In the example experiment there are no outliers for 18S 7 Repeat steps 2 through 6 for the EGR3 MAOB OGDH OSGEP and SERPING1 wells In the example experiment there are no outliers for any of the targets 6a ...

Page 195: ...amplification plot A typical amplification plot has four distinct sections a Plateau phase b Linear phase c Exponential geometric phase d Baseline IMPORTANT Experimental error such as contamination or pipetting errors can produce atypical amplification curves that can result in incorrect baseline and threshold value calculations by the 7500 software Therefore Applied Biosystems recommends that you...

Page 196: ...ase the standard deviation of the replicate groups Threshold Set Too Low The threshold is set below the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar up into the exponential phase of the curve Threshold Set Too High The threshold is set above the exponential phase of the a...

Page 197: ... or Manually adjust the baseline and or threshold see View the Analysis Settings on page 176 For More Information For more information on the Amplification Plot screen open the 7500 Software Help by clicking or pressing F1 Baseline Examples Baseline Set Correctly The amplification curve begins after the maximum baseline Baseline Set Too Low The amplification curve begins too far to the right of th...

Page 198: ...ive CT Experiments 174 Notes Publish the Data You can publish the experiment data in several ways Save the plot as an image file Print the plot Print the reaction plate layout Create slides Print a report Export data to applications such as Microsoft Excel and Microsoft PowerPoint For information on performing these procedures open the 7500 Software Help by clicking or pressing F1 ...

Page 199: ... Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Notes Section 9 2 Troubleshoot If Needed This section covers View the Analysis Settings 176 View the QC Summary 179 Omit Wells from the Analysis 181 View the Multicomponent Plot 182 View the Raw Data Plot 185 ...

Page 200: ...ettings in the 7500 software are not suitable for your experiment you can change the settings in the Analysis Settings dialog box then reanalyze your experiment About the Example Experiment In the comparative CT example experiment the default analysis settings are used without changes View the Settings 1 In the navigation pane select Analysis 2 Click Analysis Settings to open the Analysis Settings...

Page 201: ...s Select the endogenous control You can set more than one endogenous control See Set Multiple Endogenous Controls below Set the efficiency See Set Efficiency on page 178 Reject outliers Select the algorithm to use to determine RQ Min Max values confidence level or standard deviations Advanced Settings Use this tab to change baseline settings well by well Set Multiple Endogenous Controls You can se...

Page 202: ...e to produce more precise quantitation results for reactions that do not amplify with 100 efficiency By default the 7500 software calculates sample quantities based on assays that have calculated amplification efficiencies of 100 Applied Biosystems TaqMan Gene Expression Assays Inventoried and Made to Order have an amplification efficiency of 100 10 when measured over a broad dynamic range 6 logs ...

Page 203: ... QC Summary screen for any flags generated by the experiment data however no flags are generated View the QC Summary 1 In the navigation pane select Analysis QC Summary Note If no data are displayed click Analyze 2 Review the Flags Summary In the example experiment there are no flagged wells 3 In the Flag Details table look in the Frequency and Wells columns to determine the flags that appear in t...

Page 204: ...link to view information on correcting the flag You can change the flag settings Adjust the sensitivity so that more wells or fewer wells are flagged Change the flags that are applied by the 7500 software For More Information For more information on the QC Summary screen or on flag settings open the 7500 Software Help by clicking or pressing F1 Flag Description AMPNC Amplification in negative cont...

Page 205: ...easurements To ensure precision omit the outliers from the analysis About the Example Experiment In the comparative CT example experiment there are no outliers No wells need to be removed from analysis Omit Wells 1 In the navigation pane select Analysis Amplification Plot Note If no data are displayed click Analyze 2 In the Amplification Plot screen select CT vs Well in the Plot Type drop down lis...

Page 206: ...any outliers in the replicate group be sure they are flagged c Select the Omit check box next to the outlying well s 5 Click Analyze to reanalyze the experiment data with the outlying well s removed from the analysis For More Information For more information on omitting wells from the analysis open the 7500 Software Help by clicking or pressing F1 Within the Help search for the omit well topics 1 ...

Page 207: ...ct one well in the plate layout the Multicomponent Plot shows the well data Note If you select multiple wells the Multicomponent Plot screen displays the data for all selected wells simultaneously 3 In the Plot Color drop down list select Dye 4 Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend 5 View the FAM dye si...

Page 208: ...ould remain relatively constant throughout the PCR process Reporter dye The reporter dye fluorescence level should display a flat region corresponding to the baseline followed by a rapid rise in fluorescence as the amplification proceeds Any irregularities in the signal There should not be any spikes dips and or sudden changes in the fluorescence Negative control wells There should not be any ampl...

Page 209: ... you review the Raw Data Plot screen for a stable increase in signal no abrupt changes or dips from the appropriate filter View the Plot 1 In the navigation pane select Analysis Raw Data Plot Note If no data are displayed click Analyze 2 Display all 96 wells in the Raw Data Plot screen by clicking the upper left corner of the plate layout in the View Plate Layout tab 3 Click Show a legend for the ...

Page 210: ... increase occurs in signal from filter 1 which corresponds to the FAM dye filter The filters are Analysis Guidelines When you analyze your own comparative CT experiment look for the following in each filter Characteristic signal growth No abrupt changes or dips For More Information For more information on the Raw Data Plot screen open the 7500 Software Help by clicking or pressing F1 3 4 Filter 1 ...

Page 211: ... 187 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments Notes Section 9 3 Perform a Study of Multiple Experiments This section covers Section Overview 188 Design a Study 189 Analyze the Study 195 Publish the Data 202 ...

Page 212: ...name experiments Comparative Ct Study Example edm where drive is the computer hard drive on which the 7500 software is installed software name is the current version of the 7500 software Note The Comparative Ct Study Example edm file is not designed to demonstrate the use of biological replicate groups an additional example study provided with the 7500 software provides a better example You can fi...

Page 213: ...y It also describes Applied Biosystems recommended best practices as you create the study Create a New Study To create a new experiment using the Design Wizard in the 7500 software 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of the 7500 software 2 In the Home screen click Create Study Note If yo...

Page 214: ...of this chapter Complete the Study Properties Screen 1 In the navigation pane select Setup Study Properties 2 In the Study Properties pane click the Study Name field then enter Comparative Ct Study Example 3 Click the Comments field then enter Example Comparative Ct Study for the Relative Standard Curve Comparative Ct Getting Started Guide 4 Click the User Name field then enter Example User 5 In t...

Page 215: ...ycles sample volume and emulation mode IMPORTANT The 7500 software cannot combine in the same study experiments that use Fast and standard thermal cycling conditions IMPORTANT The 7500 software automatically analyzes a study after more than one experiment is added to it Consequently to ensure that the software uses the correct settings Applied Biosystems recommends that you review the analysis set...

Page 216: ...the Define Replicates screen create biological replicate groups and use them to associate samples for the analysis Biological replicates allow you to assess the representative nature of your results as they relate to the population being studied Inclusion of biological replicates can give insight into any natural variation that is present within the population About the Example Study The comparati...

Page 217: ... layout select wells D6 and D7 then click to add the technical replicate wells associated with the selected wells to the biological group The 7500 software automatically adds technical replicates of the selected wells to the biological group f Click OK 4 Select the LKBGP group that you just added to the study The 7500 software displays the details of the biological group in the Properties pane o 2...

Page 218: ...click in the plate layout to select multiple wells Click the upper left corner of the plate layout to select all 96 wells You can use the Define Replicates screen to change the name of a biological replicate group change its color identification and description and add or remove technical replicates See Edit a Biological Replicate Group below Delete an existing biological replicate group by select...

Page 219: ...e study provided with the 7500 software provides a better example You can find the alternate study file on your computer at drive Applied Biosystems software name experiments Comparative Ct Study Example Biological Groups edm Open the Example Study 1 Double click 7500 software or select Start All Programs Applied Biosystems 7500 Software software name where software name is the current version of ...

Page 220: ...roup tabs indicates the omission status of the members of the associated replicate groups For example a check mark indicates that all members of a group have been omitted from the analysis A hyphen indicates that one or more members of the group have been omitted Technical Replicates Tab Biological Replicates Tab This tab groups the results of the relative quantitation analysis by technical replic...

Page 221: ...sults Data group to evaluate the precision of the replicate groups View the Gene Expression Plot 1 In the navigation pane select Analysis Gene Expression 2 In the Gene Expression Plot screen a In the Plot Type drop down list select RQ vs Sample b In the Graph Type drop down list select Log10 c In the Orientation drop down list select Vertical Bars 3 Click Show a legend for the plot Note This is a ...

Page 222: ...licate CT values CT Mean The arithmetic average of the technical replicate CT values for the sample replicate group CT SE The sample standard deviation of the sample replicate group level CT values CT The calculated CT value for the replicate group associated with the test sample RQ The calculated relative level of gene expression for the replicate group associated with the test sample RQ Min The ...

Page 223: ... file for the example study on your computer at drive Applied Biosystems software name experiments Comparative Ct Study Example Biological Groups edm Omit Replicates from the Analysis You can use the Technical Replicates and Biological Replicates tabs of the Gene Expression screen to omit technical replicates and biological replicates from the analysis To omit a technical or biological replicate 1...

Page 224: ... that contains the well of interest 2 In the View Well Table tab select the check box in the Omit column for the well of interest IMPORTANT You cannot omit all technical replicates that belong to a reference sample belong to a reference biological group or serve as the endogenous control View the Multicomponent Plot The Multicomponent Plot displays complete spectral contribution of each dye over t...

Page 225: ... the Well Table tab then select Hide unselected data from plot to display data only from the selected rows Note You can reduce the data displayed in the plots by applying a filter using the filter function at the top of the screen See How to Simplify Data Lists Using the Filter Query on page 192 for more information on using filters Note You can use the View Well Table tab of the Multiple Plots Vi...

Page 226: ...mparison Settings group modify the comparison analysis settings a Click Edit to the right of the Comparison Analysis Settings heading b Edit the comparison analysis settings as desired then click Analyze c Click Use Comparison Analysis Settings to analyze the study data using the modified settings Note Click Use Comparison Analysis Settings to use the previous analysis settings 3 Compare the resul...

Page 227: ...ment Workflows This appendix covers Advanced Setup Workflow 204 QuickStart Workflow 206 Template Workflow 208 Export Import Workflow 210 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help ...

Page 228: ...d icon to expand the Set Up menu 3 Complete the setup screens to set up a new experiment a Click Experiment Properties default enter the experiment name then select the experiment properties b Click Plate Setup c Click Add Biological Group to create biological replicates and assign replicates to samples for each biological group in the reaction plate d Click Run Method review the reaction volume a...

Page 229: ...t will cause gaps in data collection a Load the reaction plate into the instrument b Start the run c Optional Monitor the run d Unload the reaction plate from the instrument 6 Analyze the data a Open the experiment in the 7500 software b In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example...

Page 230: ... enter the experiment name then select the experiment properties d Select the Run Method tab review the reaction volume and thermal profile then edit as needed 3 Run the experiment IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation Performing such activities while th...

Page 231: ...data a Open the experiment in the 7500 software b In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data Experiment Type Select then complete the Genotyping a Define SNP Assays and Samples tab b Assign SNP Assays and Samples tab All oth...

Page 232: ...eriment Note You can create a new experiment using the Design Wizard see Chapter 2 and Chapter 6 or Advanced Setup see page 204 3 Select File Save As Template 4 Enter a file name select a location for the template then click Save 5 Click Close Create an Experiment Using a Template 1 In the Home screen click Template Note If you do not see the Template icon click the arrow below the Design Wizard i...

Page 233: ...ill cause gaps in data collection a Load the reaction plate into the instrument b Start the run c Optional Monitor the run d Unload the reaction plate from the instrument 7 Analyze the data a Open the experiment in the 7500 software b In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example se...

Page 234: ...ort 4 Select the Export Properties tab default then a Select Sample Setup b Select One File in the drop down list c Enter a name then select a location for the export file d Select txt in the File Type drop down list IMPORTANT You cannot export xml files 5 Optional Select the Customize Export tab then select the appropriate options 6 Click Start Export 7 When prompted click Close Export Tool Creat...

Page 235: ... replace the reaction plate setup with the data from the text file Click Yes to replace the reaction plate setup 2 Use Advanced Setup to finish setting up your experiment see page 204 3 Prepare the PCR reactions 4 Run the experiment IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to e...

Page 236: ...Relative Standard Curve and Comparative CT Experiments 212 Notes 5 Analyze the data a Open the experiment in the 7500 software b In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data ...

Page 237: ...ve and Comparative CT Experiments Bibliography Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of β globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 ...

Page 238: ...Bibliography Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments 214 ...

Page 239: ...cation plot and the data are used to calculate results For genotyping or presence absence experiments fluorescence data collected during amplification are displayed in an amplification plot and the data can be used for troubleshooting amplification efficiency EFF Calculation of efficiency of the PCR amplification The amplification efficiency is calculated using the slope of the regression line in ...

Page 240: ...ach assay order The file name includes the number from the barcode on the plate The information in the AIF is provided in a tab delimited format assay mix PCR reaction component in Applied Biosystems TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays The assay mix contains primers designed to amplify a target and a TaqMan probe designed to detect amplification of the target AutoDelta I...

Page 241: ...the same cell line or tissue sample See also technical replicates blocked IPC In presence absence experiments a reaction that contains IPC blocking agent which blocks amplification of the internal positive control IPC In the 7500 software the task for the IPC target in wells that contain IPC blocking agent See also negative control blocked IPC wells CT See threshold cycle CT calibrator See referen...

Page 242: ...Concentration 10 for Reaction Mix In the 7500 software a field displayed on the Sample Dilution Calculations tab of the Reaction Setup screen For this field enter the sample concentration you want to use to add to the reaction mix for all samples in the experiment 10 for Reaction Mix indicates that the software assumes the sample or standard component of the reaction mix is at a 10 concentration F...

Page 243: ...ive control IPC In presence absence experiments a short synthetic DNA template that is added to PCR reactions You can use the IPC to distinguish between true negative results that is the target is absent in the samples and negative results caused by PCR inhibitors incorrect assay setup or reagent or instrument failure inventoried assays TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assay...

Page 244: ...ion No amplification should occur in negative control blocked IPC wells because the reaction contains no sample and amplification of the IPC is blocked Previously called no amplification control NAC negative control IPC wells In presence absence experiments wells that contain IPC template and buffer or water instead of sample Only the IPC template should amplify in negative control IPC wells becau...

Page 245: ...layed in the allelic discrimination plot and used to make allele calls In presence absence experiments fluorescence data collected during the post PCR read are displayed in the presence absence plot and used to make detection calls Also called endpoint read pre PCR read Used in genotyping and presence absence experiments the part of the instrument run that occurs before amplification The pre PCR r...

Page 246: ...results using the standard ramp speed Applied Biosystems recommends using standard reagents in your PCR reactions IMPORTANT TaqMan Fast reagents are not supported for genotyping or presence absence experiments raw data plot A plot of raw fluorescence not normalized for each optical filter reaction mix A solution that contains all components to run the PCR reaction except for the template sample st...

Page 247: ...s Measurements are normalized using the endogenous control Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates target quantity in the samples and in the reference sample The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference s...

Page 248: ...ce of quantities in the standard curve The serial factor and the starting quantity are used to calculate the standard quantity for each point in the standard curve For example if the standard curve is defined with a serial factor of 1 10 or 10 the difference between any 2 adjacent points in the curve is 10 fold series See standard dilution series slope Regression coefficient calculated from the re...

Page 249: ...The standard dilution series is prepared by serially diluting standards For example the standard stock is used to prepare the first dilution point the first dilution point is used to prepare the second dilution point and so on In the 7500 software the volumes needed to prepare a standard dilution series are calculated by the number of dilution points the number of standard replicates the starting ...

Page 250: ... The nucleic acid sequence that you want to amplify and detect target color In the 7500 software a color assigned to a target to identify the target in the plate layout and analysis plots Target Library In the 7500 software a collection of targets to add to experiments The targets in the library contain the target name reporter quencher and target color The target in the library may also contain c...

Page 251: ...t run threshold 1 In amplification plots the level of fluorescence above the baseline and within the exponential growth region The threshold can be determined automatically see automatic CT or can be set manually see manual CT 2 In presence absence experiments the level of fluorescence above which the 7500 software assigns a presence call threshold cycle CT The PCR cycle number at which the fluore...

Page 252: ...Glossary Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments 228 ...

Page 253: ...178 180 182 184 186 guidelines 80 84 90 96 101 104 106 109 110 159 165 171 177 180 182 184 186 omit wells 105 181 publish the data 174 view Amplification Plot screen 85 166 view analysis settings 100 176 view Gene Expression Plot screen 93 162 view Multicomponent Plot screen 107 182 view Multiple Plots screen 81 161 view QC Summary screen 103 179 view Raw Data Plot screen 109 185 view Standard Cur...

Page 254: ...n 198 CT experiment See comparative CT experiment CT SD column 164 Define Replicates screen 192 design experiment create new 21 113 define experiment properties 22 114 define methods and materials 24 116 finish Design Wizard 46 134 for more information 23 26 28 31 33 34 36 42 46 48 115 118 120 123 124 126 130 134 136 guidelines 23 25 28 30 32 34 36 42 46 48 115 117 120 122 124 126 130 134 136 orde...

Page 255: ...ays 42 130 Materials List screen 43 131 materials required 51 54 55 58 61 139 142 144 146 Methods Materials screen 24 116 monitor run Amplification Plot screen 72 Run Method screen 73 moving and lifting safety xvi moving parts safety xx MSDSs description xvii obtaining xvii MSDSs obtaining xi MTP flag 104 180 Multicomponent Plot screen 107 182 200 Multiple Plots screen 81 161 201 multiplex PCR 10 ...

Page 256: ...information 74 guidelines 71 monitor 72 prepare for 67 153 start 71 workflow 66 Run Method library 36 126 Run Method screen 35 125 monitor during a run 73 S safety before operating the instrument xvi biological hazards xxi chemical xvii chemical waste xix conventions xii electrical xx ergonomic xxi guidelines xviii xix instrument operation xvi moving and lifting instrument xvi moving parts xx movi...

Page 257: ...flag 104 180 threshold correct values 90 171 examples 91 172 manually adjust 101 177 training information on xi troubleshooting adjust baseline 101 177 adjust threshold 101 177 flags 104 180 omit wells 105 181 199 200 view analysis settings 100 176 202 view Multicomponent Plot screen 107 182 200 view QC Summary screen 103 179 201 view Raw Data Plot screen 109 185 U unload instrument 74 user attent...

Page 258: ...Index Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Relative Standard Curve and Comparative CT Experiments 234 ...

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Page 260: ...sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading technology and information for life scientists 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 06 2010 Part Nu...

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