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Sequence Detection Systems Chemistry Guide

1. Tissue c myc GAPDH c mycy c myCy ng Total Raji RNA ng Total Raji RNA Norm to GAPDH Rel to Brain Brain 0 031 0 618 0 05 0 038 0 532 0 07 0 032 0 521 0 06 0 038 0 550 0 07 0 032 0 577 0 06 0 037 0 532 0 07 Average 0 06 0 008 1 0 0 14 Kidney 0 365 0 049 0 35 0 338 1 035 0 33 0 423 1 042 0 41 0 334 1 086 0 31 0 334 1 021 0 33 0 372 1 139 0 33 Average 0 34 0 035 5 4 0 55 Liver 0 477 0 255 1 87 0 471 0 228 2 06 0 535 0 258 2 07 0 589 0 241 2 44 0 539 0 264 2 04 0 465 0 227 2 05 Average 2 09 0 186 33 3 2 97 Lung 0 853 0 085 0 97 0 900 0 084 0 88 0 956 0 082 1 00 0 900 0 093 0 87 0 996 0 112 0 87 0 859 0 090 0 84 Average 0 90 0 062 14 4 0 99 3 42 Sequence Detection Systems Chemistry Guide How to Perform Multiplex PCR with the Comparative C Method Example of Multiplex PCR with the Comparative Cz Method Multiplex PCR Same Tube Method for Relative Quantification To perform multiplex PCR with the comparative C method for relative quantification Perform a validation experiment Perform a run on your SDS instrument This includes Setting up a reaction plate Analyzing the data Determine the AC value Perform the AAC calculation See the example below for an illustration of these steps This example illustrates the use of multiplex PCR with the comparative C method for relative quantific
2. For more information on absolute and relative quantification refer to Livak and Schmittgen 2001 This document contains detailed procedures for performing the experiments referenced in this section Sequence Detection Systems Chemistry Guide Relative Standard Curve Method for Quantification Relative Standard Curve Method for Quantification It is easy to prepare standard curves for relative quantification because quantity is expressed relative to some basis sample such as the calibrator For all experimental samples target quantity is determined from the standard curve and divided by the target quantity of the calibrator Thus the calibrator becomes the 1x sample and all other quantities are expressed as an n fold difference relative to the calibrator For example in a study of drug effects on expression the untreated control would be an appropriate calibrator Requirements The requirements below are critical for proper use of the relative standard curve method for quantification tis important that stock RNA or DNA be accurately diluted but the units used to express this dilution are irrelevant If two fold dilutions of a total RNA preparation from a control cell line are used to construct a standard curve the units could be the dilution values 1 0 5 0 25 0 125 and so on By using the same stock RNA or DNA to prepare standard curves for multiple plates the relative quantities determined can be compared across the
3. 0 000 cee eee 3 22 Primer Limiting in Multiplex Assays eee eee 3 23 Section 3 2 Selecting a Data Analysis Approach and Determining Results 3 25 In This Sectloni i nr cua cos hie ete eee ME Sain Sar aerated RE ANE 3 25 Data Analysis ise eta ioe eee a ate ee aie Sea an a mos qon RE erbe nns 3 26 General Process a vis help eee EA RG Set et ee dea EE 3 26 Resources for Data Analysis lille 3 26 Relative or Absolute Quantification ssas aeaaaee eee 3 26 What Is Relative Quantification 0 0 0 0 eee 3 26 Calculation Methods for Relative Quantification 0000005 3 27 What Is Absolute Quantification 2000 0 eee 3 27 Calculation Methods for Absolute Quantification 0000000 3 27 TetmsAJSed oc ce oes eee teet s ber a a ate EC Se ee 3 28 For More Information lleeeeeeeee RR nn 3 28 Relative Standard Curve Method for Quantification 0000000e 3 29 Requirements une Sea eb ewe cate eee a arena ARE RR EUR EDU 3 29 Endogenous Control occ sss so gomedeD pas ER MEd ee eee bee alot 3 29 Standards 4 sire ERI ih ce te gees ps RC Ge GD eae a A 3 29 How to Perform the Relative Standard Curve Method 3 30 Example of the Relative Standard Curve Method lues 3 30 Comparative CT Method for Relative Quantification 2000000 3 35 Formula ranae e Seka RAE dente oad dece ens S NT ER 3 35 Relati
4. Make sure the kits have been stored properly per instructions on the packaging and have not expired Interactions between primers and probe with the passive reference dye that cannot be compensated for by the baseline subtraction Redesign the primers and or probe Standard curve poor slope Note A slope value of 3 32 is equal to approximately 100 efficiency Assay design guidelines not followed Follow guidelines precisely Optimize assay probe concentrations Incorrect dilutions Redilute the samples Ensure pipettes are calibrated Inhibitors present in the reaction Verify extraction method and reprecipitate DNA or RNA Improper reaction conditions protocols must be followed precisely Follow Applied Biosystems recommended thermal profile 6 4 Sequence Detection Systems Chemistry Guide Troubleshooting Quantification Assays Table 6 2 Troubleshooting chemistry problems quantification assays continued Observation Possible Cause s Recommended Action Standard curve bad correlation Incorrect baseline and Verify settings according to the user guide coefficient threshold settings specific to your system Note The best correlation Improper pipetting Check calibration of the pipettes Pipette more coefficient is 1 0 than 5 uL of sample Incorrect dilutions Redilute the samples Sequence Detection Systems Chemistry Guide 6 5 Chap
5. 3 15 Optimizing Primer Concentrations 0 00 cece eee eee 3 17 Optimizing the Probe Concentration 2 0 0 2 e eee eee eee 3 19 Using Multiplex PCR cca gov stude e rh Rete d oe eda ee et 3 22 Sequence Detection Systems Chemistry Guide 3 3 Chapter 3 Gene Expression and Other Quantitative Assays About Quantitative Assays What Is a Quantification Assay Instruments Terms Used in Quantification Analysis 3 4 A Quantification Assay is a real time PCR assay It measures the amount of a nucleic acid target during each amplification cycle of the PCR The target may be DNA cDNA or RNA There are three types of Quantification Assays discussed in this chemistry guide DNA cDNA quantification RNA quantification using one step reverse transcription polymerase chain reaction RT PCR RNA quantification using two step RT PCR Note For more information on one step and two step RT PCR see About Real Time PCR Assays on page 1 3 Quantification Assays can be used with the following Sequence Detection System SDS instruments Applied Biosystems 7900HT Real Time PCR System 7900HT System Applied Biosystems 7300 Real Time PCR System 7300 System Applied Biosystems 7500 Real Time PCR System 7300 System ABI PRISM 7000 Sequence Detection System 7000 System ABI PRISM 7700 Sequence Detection System 7700 System GeneAmp 5700 Sequence Detection System 5700 System IMPO
6. Master Mix No AmpErase UNG Assays on Demand Assays on Demand SNP 4331183 Genotyping Products Assays by Design Assays by Design Service Please see our web site for part for SNP Assays numbers Keyword genomic assays See How to Obtain Support on page xi Sequence Detection Systems Chemistry Guide Designing Your Own Allelic Assay Quantify the amount of genomic DNA in samples before using Assays On Demand SNP Genotyping Products Generate a standard curve using the DNA Kit PN 401970 and the RNase P gene primers and probe provided in the TaqMan RNase P Detection Reagents Kit PN 4316831 Note TaqMan RNase P Detection Reagents Kit uses a TaqMan probe with TAMRA dye as the quencher Using the Universal Thermal Cycling Parameters Thermal Cycling Parameters Allelic discrimination assays designed using Applied Biosystems Assay Design Guidelines can be run using the same universal thermal cycling parameters This eliminates any optimization of the thermal cycling parameters and means that multiple assays can be run on the same plate without sacrificing performance The thermal cycling parameters listed in Table 4 4 are recommended for allelic discrimination assays Table 4 4 Thermal cycling parameters for allelic discrimination assays Times and Temperatures Initial Step PCR Each of 40 Cycles AmpliTaq Gold AmpErase UNG DNA Polymerase Man aione Activation Melt Anneal Extend HO
7. Q quantification absolute 3 27 calculating absolute 3 27 calculating relative 3 27 relative 3 26 relative formula 3 35 relative method choice 3 27 relative vs absolute 3 26 standard curve requirements 3 29 terms defined 3 28 validation 3 35 quantification analysis terms defined 3 4 quantification assay 5 nuclease 3 17 category 1 2 design guidelines 3 9 explained 3 4 instruments used 3 4 optimizing SYBR Green I Dye 3 18 reagent configurations 3 13 selecting chemistry 3 6 troubleshooting analyzed run data 6 2 troubleshooting chemistry problems 6 3 with SYBR Green I dye 1 6 R radioactive waste handling xvii reagent configurations allelic configuration assays 4 8 selecting 3 13 selection 4 8 real time PCR defined 1 3 quantification process 3 6 TaqMan detection process 2 3 recommendations probe concentration 3 20 thermal cycling parameters 3 15 relative quantification and multiplex PCR 3 40 calculation methods 3 27 comparitive CT 3 35 comparitive CT formula 3 35 relative standard curve example 3 30 multiplex PCR 3 41 performing 3 30 relative values 3 31 Index 3 relative values relative standard curve 3 31 reporter dyes multiplex 3 40 requirements dye binding 2 2 RNA one step thermal cycling parameters 3 15 RNA quantification and one step RT PCR 3 13 and two step RT PCR thermal cycling parameters 3 15 RNA two step thermal cycling parameters 3 16 rRNA primers and multiplex assays 3 23 RT PCR
8. The guidelines below are critical for proper use of the absolute standard curve method for quantification tis important that the DNA or RNA be a single pure species For example plasmid DNA prepared from coli often is contaminated with RNA which increases the A measurement and inflates the copy number determined for the plasmid Accurate pipetting 1s required because the standards must be diluted over several orders of magnitude Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A value This concentrated DNA or RNA must then be diluted 10 to 10 fold to be at a concentration similar to the target in biological samples The stability of the diluted standards must be considered especially for RNA Divide diluted standards into small aliquots store at 80 C and thaw only once before use An example of the effort required to generate trustworthy standards is provided by Collins Collins et al 1995 who report on the steps they used in developing an absolute RNA standard for viral RNA quantification tis generally not possible to use DNA as a standard for absolute quantification of RNA because there is no control for the efficiency of the reverse transcription step The absolute quantities of the standards must first be known by some independent means Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute standards Concentration is measured b
9. To perform the validation experiment continued 6 Plot the log of the input amount vs AC see the figure below Relative Efficiency Plot p53 vs GAPDH y 0 0034x 4 7295 R 0 0003 1 0 log ng Total RNA As a guideline the absolute value of the slope of log input amount vs AC should less than 0 1 The the slope in the above figure is 0 0034 which passes this test Note The less than 0 1 guideline should provide gene expression results with a low degree of experimental variation However if it is essential to quantify any variation introduced by the comparative C method parallel experiments using the comparative Cz method and the standard curve method should be performed and the results compared If the efficiencies of the two systems are Then 0 1 You can use the AAC calculation for the relative quantification of target without running standard curves on the same plate 0 1 Perform the validation experiment over a larger dynamic range 5 to 6 logs or design and synthesize new primers to improve efficiency Perform the quantification using the standard curve method Sequence Detection Systems Chemistry Guide Comparative CT Method for Relative Quantification Table 3 15 Average C value for c myc and GAPDH at different input amounts Input Amount c myc GAPDH AC ng Total RNA Average C4 Average C4 c m
10. skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves N py elsasg ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions Sequence Detection Systems Chemistry Guide General Instrument Safety General Instrument Safety gt Enc PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Chemical Safety Chemical Hazard Warning About MSDSs Obtaining MSDSs NSE CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions T VUAN CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument Pa EN CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover faste
11. N808 0235 TaqMan EZ RT PCR Kit 200 1000 Units rTth DNA Polymerase 100 Units AmpErase UNG dUTP dATP dCTP dGTP TaqMan EZ Buffer Mn OAc 100 GAPDH control reactions and protocol Sequence Detection Systems Chemistry Guide C 7 Appendix C Part Numbers Sequence Detection Control Reagents Part Number Kit Number of Reactions 4308329 TaqMan Ribosomal RNA Control Reagents VIC Dye 1000 Human Control RNA rRNA Probe VIC rRNA Forward Primer and rRNA Reverse Primer 4308310 TaqMan Ribosomal RNA Control Reagents Protocol VIC Dye 4316844 TagMan RNase P Control Reagents VIC Dye 1000 20X primer and probe VIC mix and Human Genomic Control DNA 4308313 TaqMan Rodent GAPDH Control Reagents VIC Dye 1000 Rodent Control RNA rodent GAPDH Probe VIC rodent GAPDH Forward Primer and rodent GAPDH Reverse Primer 4308318 TaqMan Rodent GAPDH Control Reagents Protocol VIC Dye 402869 TagMan GAPDH Control Reagents Human 100 Human Control RNA GAPDH Probe JOE GAPDH Forward Primer and GAPDH Reverse Primer 401846 TagMan p actin Detection Reagents 100 401970 TagMan DNA Template Reagents 4308323 TaqMan Exogenous Internal Positive Control Reagents VIC Dye 200 10X exogenous IPC primer and probe VIC mix 10X exogenous IPC blocking reagent and 50X exogenous IPC target Kit represents an IPC assay optimized with TaqMan Universal PC
12. on page 3 9 and Designing Your Own Allelic Assay on page 4 6 Important Design Steps Applied Biosystems Assay Design Guidelines contain the following important steps Designing primers and probes using Primer Express software The Primer Express software is used to design primers and probes The software uses a set of default parameters to automatically select primer and probe sets Selecting the appropriate reagent configuration There are several TaqMan and SYBR Green I dye chemistry kits available The reagent configuration you use will depend on your assay type For quantification assays see Selecting the Appropriate Reagent Configuration on page 3 13 Using universal thermal cycling parameters All assay types designed using Applied Biosystems Assay Design Guidelines can be run using universal thermal cycling parameters This eliminates any optimization of the thermal cycling parameters and allows multiple assays to be run on the same plate without sacrificing performance Using default primer and probe concentrations or optimizing primer and probe concentrations When you use Applied Biosystems Assay Design Guidelines you can use default primer and probe concentrations for non multiplex optimized assays or you can optimize primer and probe concentrations For quantification assays see Optimizing Primer Concentrations on page 3 17 and Optimizing the Probe Concentration on page 3 19 IMPORTANT The
13. 3 17 Chapter 3 Gene Expression and Other Quantitative Assays SYBR Green Dye Quantifica tion Assays 2 a Linear view Tae Plot Group A lt Plot Group B 000 E1 Plot Group C 3 000 61 Cycle b Log view Plot Group A Plot Group B 1 000 Plot Group C g a4 lius 1000E2 e XL NN LAL 5 10 15 20 25 30 35 40 Cycle Figure 3 2 Primer optimization experimental results showing amplification plots of primer combinations Plot group key A Combinations that contain at least 300 nM of forward and reverse primer B Combinations that contain at least 150 nM of forward and reverse primer C Combinations that contain at least 50 nM of forward and reverse primer Optimizing primer concentrations is slightly more complex for a SYBR Green I dye Quantification Assay The same primer optimization matrix should be performed however this time it must include NTCs In this case the primer concentrations selected should provide a low Cy and high AR when run against the target template but should not produce nonspecific product formation with NTCs An ideal NTC amplification plot is shown in Figure 3 1 on page 3 6 Dissociation curves or gel analysis can be extremely useful when selecting optimal primer concentrations for a SYBR Green I dye Quantification Assay This is demonstrated in Figure 3 3 on page 3 19 which shows the results from a primer optimization matrix at
14. 4331182 consist of target assays Products and endogenous control assays to be used in a 5 fluorogenic nuclease assay to amplify and detect expression of specific nucleic acid sequences The latest information on available products and specific product uses is on the Applied Biosystems web site See How to Obtain Support on page xi For more information about ordering Assays on Demand Gene Expression Products contact your Applied Biosystems representative About Target Target assays from the Assays on Demand Gene Expression Products possess the Assays following characteristics A variety of Assays on Demand Gene Expression Products are available for detecting transcripts corresponding to many biological pathways Many of the target assays are specifically designed to detect and quantify cDNA sequences without detecting genomic DNA Target assays contain TaqMan MGB probes FAM dye labeled combined with primers at non limiting concentrations Assays by Design Service If a particular gene expression assay or SNP of interest is not available as an Assays on Demand product Applied Biosystems Assays by Design service can design an assay based on sequence information you submit The Assays by Design service see Selecting an Assay Source on page 1 7 is an assay development service that designs synthesizes formulates and delivers analytically quality controlled primers and probes for gene expression assays For more info
15. 95 C 15sec 9 95 C 1min 60 C IMPORTANT For most applications and when large amounts of cDNA are required Applied Biosystems recommends 120 minutes at 37 C for reverse transcription to achieve optimal conversion Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay Optimizing Primer Concentrations By independently varying forward and reverse primer concentrations you can identify the concentrations that provide optimal assay performance Primers are always in large molar excess during the exponential phase of PCR amplification by adjusting their initial concentration their effective melting temperatures can be adjusted When using Universal TaqMan PCR Master Mix Applied Biosystems recommends the primer concentrations shown in Table 3 11 Detailed discussions follow for the Primer optimization matrix page 3 17 5 Nuclease quantification assays using TaqMan Probe based chemistry page 3 17 SYBR Green I Dye quantification assays page 3 18 Default Primer The recommended primer concentrations listed in Table 3 11 are for DNA and Concentrations cDNA Quantification Assays Table 3 11 Recommended primer concentrations for DNA and cDNA quantification assays Concentrations nM Chemistry Forward Primer Reverse Primer TaqMan probe 900 900 SYBR Green dye 50 50 Primer A primer optimization matrix allows you to determine the minimum primer Optim
16. Allele 2 probe A minor groove binder MGB This modification increases the melting temperature T p without increasing probe length Afonina et al 1997 Kutyavin et al 1997 which allows the design of shorter probes This results in greater differences in T values between matched and mismatched probes which produces more accurate allelic discrimination A nonfluorescent quencher NFQ at the 3 end of the probe Because the quencher does not fluoresce Applied Biosystems sequence detection systems can measure reporter dye contributions more accurately During PCR each probe anneals specifically to complementary sequences between the forward and reverse primer sites AmpliTaq Gold DNA polymerase can cleave only probes that hybridize to the allele sequence Cleavage separates the reporter dye from the quencher dye which results in increased fluorescence by the reporter dye Thus the fluorescence signal s generated by PCR amplification indicate s the alleles that are present in the sample Mismatches Between Probe and Allele Sequences Mismatches between a probe and allele reduce the efficiency of probe hybridization Furthermore AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe rather than cleave it to release reporter dye Sequence Detection Systems Chemistry Guide 4 3 Chapter 4 Allelic Discrimination Assays The figure below illustrates results from matches and mismatches between allele and
17. Expression FAM Nonfluorescent minor quencher groove binder SNP Genotyping FAM VIC Nonfluorescent minor quencher groove binder Assays by Design TaqMan MGB Gene Expression FAM Nonfluorescent minor quencher groove binder SNP Genotyping FAM VIC Nonfluorescent minor groove binder TaqMan MGB Probes Recommended Applied Biosystems recommends the general use of TaqMan MGB probes especially when conventional TaqMan probes exceed 30 nucleotides The TaqMan MGB probes contain A nonfluorescent quencher at the 3 end Allows the Real Time PCR instruments to measure the reporter dye contributions more precisely because the quencher does not fluoresce A minor groove binder at the 3 end Increases the melting temperature T p of probes Afonina et al 1997 Kutyavin et al 1997 allowing the use of shorter probes Consequently the TaqMan MGB probes exhibit greater differences in T values between matched and mismatched probes which provides more accurate allelic discrimination 2 4 Sequence Detection Systems Chemistry Guide Selecting the Appropriate Chemistry Selecting the Appropriate Chemistry The SYBR Green I dye and TaqMan Probe based chemistry can be used for the assay types listed in Table 2 2 See Table 2 3 for considerations for choosing SYBR Green or TaqMan Probe based chemistry Table 2 2 Assay type using SYBR Green 1 dye or TaqMan Probe based chemistry Assay Type TEE Alleli
18. For information see Primer Design Using Primer Express Software on page 4 8 The TaqMan MGB probes are two modified conventional TaqMan probes One probe matches the Allele 1 sequence One probe matches the Allele 2 sequence Sequence Detection Systems Chemistry Guide When to Use TaqMan MGB Probes Allelic Discrimina tion Probe Design Guidelines Designing Your Own Allelic Assay Table 4 2 below summarizes the TaqMan MGB probe features Table 4 2 Taqman MGB probe features Probe 5 Label 3 Label Other Features TaqMan MGB FAM or VIC Nonfluorescent minor groove binder dye quencher Applied Biosystems recommends the general use of TaqMan MGB probes for allelic discrimination assays To achieve estimated T values of 65 to 67 C using probes shorter than 20 nucleotides To obtain greater differences in T values between matched and mismatched probes than with conventional TaqMan probes To obtain more precise measurements of dye contributions IMPORTANT When designing probes it is important to consider probes from both strands To label allelic discrimination probes use VIC or FAM dyes Avoid probes with G residue at the 5 end of the probe A G residue adjacent to the reporter dye will quench the reporter fluorescence even after cleavage Select probes using Primer Express software estimated T of 65 to 67 C Make TaqMan MGB probes as short as possible with
19. Polymerase Melt Anneal Extend P Activation HOLD HOLD 40 CYCLES 30 min 48 C 10 min 95 C 15 sec 95 C 1 min 60 C a Not applicable for the TaqMan EZ RT PCR Kit Protocol See the TaqMan EZ RT PCR Kit Protocol for the appropriate values Chapter 3 Gene Expression and Other Quantitative Assays RNA Quantification Using Two Step RT PCR The thermal cycling parameters listed in Table 3 9 and Table 3 10 are recommended for RNA Quantification Assays using two step RT PCR The parameters apply to both TaqMan Probe based and SYBR Green I dye chemistries Table 3 9 Thermal cycling parameters for RNA quantification assays using two step RT PCR Times and Temperatures HOLD HOLD HOLD 1 RT Step 10 min Q 25 C 30 min 48 C 5 min 95 C Initial Steps PCR Each of 40 Cycles AmpliTaq Gold NR DNA wich Anneal dde Polymerase Extend 2 PCR St PCR Step Activation Activation HOLD HOLD CYCLE 2 min Q 50 C 10min Q 95 C 15sec Q9 95 C 1 min 60 C Table 3 10 Thermal cycling parameters for RNA Quantification assays using the high capacity cDNA archive kit when using two step RT PCR Times and Temperatures HOLD HOLD 1 RT Step Step 1 Step 2 10 min 25 C 120 min 37 C Initial Steps PCR Each of 40 Cycles S NOT NAT Melt Anneal 2 PCR Step Activation Polymerase Extend HOLD HOLD CYCLE 2min 9 50 C 10 min 9
20. R 1992 Simultaneous amplification and detection of specific DNA sequences Biotechnology 10 413 417 Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR Real time monitoring of DNA amplification reactions Biotechnology 11 1026 1030 Kutyavin I V Lukhtanov E A Gamper H B and Meyer R B 1997 Oligonucleotides with conjugated dihydropyrroloindole tripeptides base composition and backbone effects on hybridization Nucleic Acids Res 25 3718 3723 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lee L G Connell C R and Block W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucleic Acids Res 21 3761 3766 Livak K J and Schmittgen T D 2001 Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 T Method Methods 25 402 408 Livak K J Flood S J A Marmaro J Guisti W Deetz K 1995 Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization PCR Methods Appl 4 357 62 Livak K J Flood S J A Marmaro J and Mullah K B inventors Perkin Elmer Corporation Foster City CA assignee 2 Mar 1999 Hybridization assay using self quenching fluorescence probe United States patent 5 876 930 Livak K J Marmaro J and Todd J A 1995 Towards fully automated genome wide polymor
21. Rel to Brain Brain 32 38 25 07 7 31 32 08 25 29 6 79 32 35 25 32 7 03 32 08 25 24 6 84 32 34 25 17 7 17 32 13 25 29 6 84 Average 6 93 0 16 0 00 0 16 1 0 0 9 to 1 1 Kidney 28 73 24 30 4 43 28 84 24 32 4 52 28 51 24 31 4 20 28 86 24 25 4 61 28 86 24 34 4 52 28 70 24 18 4 52 Average 4 47x0 14 2 47x0 14 5 5 5 0 to 6 1 Liver 28 33 26 36 1 97 28 35 26 52 1 83 28 16 26 34 1 82 28 02 26 44 1 58 28 15 26 31 1 84 28 37 26 53 1 84 Average 1 81 0 13 5 12x0 13 34 8 31 9 to 38 0 3 44 Sequence Detection Systems Chemistry Guide Multiplex PCR Same Tube Method for Relative Quantification Table 3 18 Relative quantification using multiplex PCR with the comparative C method continued AC BST c mycy Tissue c myc Cy GAPDH C4 c myc GAPDH ACT r AC Rel to Brain Lung 27 47 24 55 2 92 27 39 24 33 3 06 27 30 24 43 2 87 27 39 24 32 3 07 27 24 24 18 3 06 27 46 24 34 3 12 Average 3 02 0 10 3 92 0 10 15 1 14 1 to 16 2 Sequence Detection Systems Chemistry Guide 3 45 Chapter 3 Gene Expression and Other Quantitative Assays Standard Curve Method for Absolute Quantification Requirements Standards How to Perform the Absolute Standard Curve Method The standard curve method for absolute quantification is similar to the relative standard curve method except the absolute quantities of the standards must first be known by some independent means
22. Running both assays in a single tube reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes Sequence Detection Systems Chemistry Guide 1 5 Chapter 1 Introduction Selecting the Sequence Detection Chemistry TagMan Probe Based Chemistry SYBR Green Dye Chemistry Applied Biosystems has developed two types of chemistries that can be used to detect PCR products on the SDS instruments e TaqMan Probe based fluorogenic 5 nuclease chemistry SYBR Green dye chemistry These chemistries are discussed in detail in Chapter 2 Applied Biosystems TaqMan Probe based chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles The Applied Biosystems patented fluorogenic probe design which incorporates the reporter dye on the 5 end and the quencher dye on the 3 end has greatly simplified the design and synthesis of effective 5 fluorogenic nuclease assay probes Livak Flood et al 1995 Assay Types that Use TaqMan Probe Based Chemistry The TaqMan Probe based chemistry can be used for the following assay types Quantification including One step RT PCR for RNA quantification Two step RT PCR for RNA quantification DNA quantification Allelic Discrimination Plus Minus The SYBR Green I dye chemistry uses SYBR Green I dye a double stranded DNA binding dye to detect PCR products
23. The following are Assays by Design service advantages Use of the service allows researchers to save the time money and effort involved in assay design synthesis and formulation of the gene specific assays SNP genotyping assays are tested to reduce the need to test assays in your laboratory saving you time and labor Sequence Detection Systems Chemistry Guide 1 7 Chapter 1 Introduction Designing Your Own Assay Cost per data point is less than the cost of materials and time to build your own Assays are delivered ready to use dilute the concentrated assay mix add TaqMan Universal PCR Master Mix and cDNA and load on an ABI PRISM Sequence Detection System platform of your choice For more information contact your Applied Biosystems representative When designing your own assay follow the Assay Design Guidelines provided in this document These simple easy to follow guidelines have been developed by Applied Biosystems to optimize results when using SDS instruments and TaqMan probes or SYBR Green I dye chemistries Note Applied Biosystems Assay Design Guidelines do not guarantee that all assays will provide the same level of performance and sensitivity Even the most scrupulous design parameters cannot account for all the possible variables that can exist between two different assay systems Guidelines for quantification and allelic discrimination are detailed in Designing Your Own Quantification Assay
24. and Determining Results 3 25 Data Analysis cei eats idea act SA ea Re atts ee fed ae Mina ade tate te 3 26 Relative or Absolute Quantification 0 0 0 0 0 0 ccc ee ee eens 3 26 Relative Standard Curve Method for Quantification 0005 3 29 Comparative CT Method for Relative Quantification 3 35 Multiplex PCR Same Tube Method for Relative Quantification 3 40 Standard Curve Method for Absolute Quantification 0 08 3 46 Sequence Detection Systems Chemistry Guide 3 1 Chapter 3 Gene Expression and Other Quantitative Assays 3 2 Sequence Detection Systems Chemistry Guide Section 3 1 In This Section Section 3 1 Introduction Introduction The following topics are covered in this section Section 3 1 Introduction 0 cece ccc cece ee eee eee cece ee eees 3 3 About Quantitative Assays esee 3 4 Selecting a Quantification Assay Chemistry 0000 cece cece eee 3 6 Purchasing a Preformulated or Custom Designed Quantification Assay 3 7 Assays on Demand Gene Expression Product 0200 eee 3 7 Assays by Design Service 0 0c ccc cee eens 3 8 Designing Your Own Quantification Assay 2 0 0 e eee eee eee eee 3 9 Primer and Probe Design Using Primer Express Software 3 10 Selecting the Appropriate Reagent Configuration 3 13 Using the Universal Thermal Cycling Parameters
25. as they accumulate during PCR cycles An important difference between the TaqMan probes and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry will detect all double stranded DNA including nonspecific reaction products A well optimized reaction is therefore essential for accurate results You can not perform multiplex PCR using SYBR Green I dye Assay Types that Use SYBR Green Dye Chemistry The SYBR Green I dye chemistry can be used for quantification assay types including One step RT PCR for RNA quantification Two step RT PCR for RNA quantification DNA quantification Sequence Detection Systems Chemistry Guide Selecting an Assay Source Selecting an Assay Source After you select an assay type and sequence detection chemistry you can Purchase a predesigned assay Assays on Demand Products Have Applied Biosystems custom design an assay to your specifications Assays by Design M Service Design your own assay of TaqMan probe and primers using Primer Express software Note Assays on Demand Product and Assays by Design Service products are intended for use in singleplex reactions Assays on Assays on Demand products provide the most comprehensive collection of Demand biologically informative predesigned quality controlled and validated assays ready Products touse on an ABI PRISM Sequence Detection System Assays on Demand Products include Assays on Demand SNP Genotyping
26. contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup Never bring amplified PCR products into the PCR setup area PCR amplification Analysis of PCR products Open and close all sample tubes carefully Avoid splashing or spraying PCR samples Use positive displacement or air displacement pipettors with filter plugged tips Change tips after each use Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 10 bleach solution or 70 ethanol Sequence Detection Systems Chemistry Guide 2 7 Chapter 2 Chemistry Overview 2 8 Sequence Detection Systems Chemistry Guide Gene Expression and Other Quantitative Assays Section 3 1 Introduction 0 cc cece cc cece ee eee een 3 3 About Quantitative Assays sese 3 4 Selecting a Quantification Assay Chemistry 00 cece cece eee 3 6 Purchasing a Preformulated or Custom Designed Quantification Assay 3 7 Assays on Demand Gene Expression Product 7 Assays by Design Service 8 Designing Your Own Quantification Assay 0 e cece eee eee 3 9 Primer and Probe Design Using Primer Express Software 10 Selecting the Appropriate Reagent Configuration 13 Using the Universal Thermal Cycling Parameters 15 Optimizing Primer Concentrations 17 Optimizing the Probe Concentration 19 Using Multiplex PCR 22 Section 3 2 Selecting a Data Analysis Approach
27. delta CT determining 3 38 performing calculation 3 38 design guidelines assay 1 8 primer and probe 3 12 quantification assays 3 9 disposables sequence detection C 11 DNA cDNA quantification thermal cycling parameters 3 15 documentation feedback x related to this guide x dye binding methods of 2 2 requirements for real time PCR 2 2 SYBR Green I dye 2 2 E efficiency arget and reference 3 37 endogenous control and standardizing 3 29 endpoint assay allelic discrimination 4 2 category 1 2 described 1 5 Index 2 G G C content and amplicon sites 3 11 gene expression data analysis process 3 26 guidelines chemical safety xvi chemical waste disposal xvi chemical waste safety xvii guidelines designing an assay 1 8 H hairpin loops and primer choice 3 7 hazard symbols See safety symbols on instruments hazards See safety IMPORTANT description xiv IMPORTANTS description ix instruments sequence detection C 2 internal positive control plus minus assay 5 2 IPC incorporating 5 3 plus minus assay 5 2 italic text when to use ix K kits sequence detection PCR reagent C 4 sequence detection RT PCR C 6 sequencedetection reaction controls C 7 sequence detection C 3 M melting temperature and amplicon sites 3 11 menu commands conventions for describing ix mismatch in allelic discrimination assay 4 3 MSDSs description xv obtaining xv MSDSs obtaining xi multicomponenting 3 40 m
28. dye primer optimization should be bypassed only with caution However if all guidelines are followed concentrations of 50 nM forward and reverse primer should provide robust amplification with a good level of specificity when using DNA or cDNA as a template This assumption should however always be verified by checking for nonspecific product formation with either dissociation curve or gel analysis Asa general rule a 5 nuclease quantification assay should enable detection and accurate quantification down to less than 50 copies of a target sequence with even greater sensitivity possible ASYBR Green I dye Quantification Assay is capable of similar performance however nonspecific product formation can potentially increase the minimum detection limit Sequence Detection Systems Chemistry Guide 3 9 Chapter 3 Gene Expression and Other Quantitative Assays Primer and Probe Design Using Primer Express Software Selecting an Amplicon Site for Gene Expression Assays About the Primer and Probe Design Guidelines The Primer Express software uses recommended parameters to select primers and probes based on the DNA sequence that you provide If you are designing your own assay follow the summary of the primer and probe design guidelines for Quantitative Assays shown in Table 3 3 on page 3 12 For a detailed discussion of these guidelines see About the Primer and Probe Design Guidelines below Note Even though no probe i
29. eee 3 8 Designing Your Own Quantification Assay 00 cee ee 3 9 Important Design Steps 0 000 eee 3 9 CONCIUSIONS c 3 9 Primer and Probe Design Using Primer Express Software 3 10 Selecting an Amplicon Site for Gene Expression Assays 3 10 About the Primer and Probe Design Guidelines lees 3 10 Summary of Primer and MGB Probe Design Guidelines 3 12 Selecting the Appropriate Reagent Configuration 0 eee eee 3 13 Recommended Reagent Configurations 00 e eee ees 3 13 About Universal Master Mix Reagents 2 00 e eee eee eee ees 3 14 About the Reagent Components 2 000 c eee ee 3 14 Using the Universal Thermal Cycling Parameters 00 0 eee eae 3 15 Recommended Thermal Cycling Parameters 00 aaua eee ee eee 3 15 Optimizing Primer Concentrations 0 0 0 c eee eee 3 17 Default Primer Concentrations 0 eee eee 3 17 Primer Optimization Matrix 00 000 cee es 3 17 5 Nuclease Quantification Assays 0 6 0 0 eee 3 17 SYBR Green Dye Quantification Assays saasaa aeaaea 3 18 Sequence Detection Systems Chemistry Guide Optimizing the Probe Concentration 0 0 00 ee eee 3 19 Recommended Probe Concentrations 0 000 eee eee eee 3 20 Using Multiplex PCR s 2 visse REED RR Vedas Soe alae ede 3 22 Multiplex in Contrast to Singleplex
30. endogenous control are amplified in separate tubes Dilutions of a cDNA sample prepared from total Raji RNA are used to construct standard curves for the c myc and the GAPDH amplifications The unknown samples samples to characterize are cDNA prepared from total RNA isolated from human brain kidney liver and lung Performing the Run The procedure below is a general outline for performing a run Refer to your instrument s user manual for detailed instructions Set up a reaction plate Place the reaction plate on your SDS instrument and start the run When the run is complete analyze the data Set the threshold and create a standard curve from the data The figure below shows the standard curve for the amplification of the c myc target detected using a FAM dye labeled probe c Myc Standard Curve y 3 385x 25 712 R 0 9979 1 5 1 0 5 Log ng Total RNA Sequence Detection Systems Chemistry Guide Relative Standard Curve Method for Quantification Determining the Relative Values To determine the relative value l Calculate the log input amount by entering the following formula in one cell of the work sheet of any spreadsheet program cell containing Cr value b m where b y intercept of standard curve line and m slope of standard curve line Note In this example b 25 712 and m 3 385 for the equation y mx b Calculate t
31. not change with experimental conditions or across samples may serve as an endogenous control Therefore the more targets you have in a singleplex format the higher the probability that you will have one or more suitable endogenous controls against which to normalize your remaining targets 3 22 Sequence Detection Systems Chemistry Guide Primer Limiting in Multiplex Assays Designing Your Own Quantification Assay To generate an accurate multiplex assay it is important to ensure that the amplification of one species does not dominate the other Otherwise the amplification of a highly abundant species can prevent the less abundant species from amplifying efficiently Such a scenario could easily produce inaccurate results and in severe cases inhibit detection of the less abundant species completely This situation can be avoided by limiting the concentrations of the primers used to amplify the more abundant species thereby turning off the amplification soon after the Cy has been established Primer limitation results in the reaction components common to both assays not being exhausted allowing the amplification of the less abundant species to continue at high efficiency If the more abundant species is not known it should be determined before entering into a multiplex assay system by running both targets in separate tubes Both amplifications should be primer limited if neither species is consistently more abundant Considering Re
32. obtain results Assay Contents Each SNP genotyping assay consists of One tube containing 20X SNP Genotyping Assay Mix sufficient for 750 uL reactions CD ROM containing assay information and PDFs of the protocol and product insert About SNP Genotyping Assay Mix The SNP Genotyping Assay Mix contains Sequence specific forward and reverse primers to amplify the SNP of interest Two TaqMan MGB probes One probe labeled with VIC dye detects the Allele 1 sequence One probe labeled with FAM dye detects the Allele 2 sequence About the Assay Information The assay information consists of Genomic information about the SNP including the chromosomal location allele frequency and context sequence Information about the packaging of each assay tube including the location in the plate rack and the 2 D bar code Sequence Detection Systems Chemistry Guide 4 5 Chapter 4 Allelic Discrimination Assays Assays by Design Service If a particular SNP assay of interest is not available as an Assays on Demand product you can use Applied Biosystems Assays by Design service see Chapter 1 The Assays by Design service is an assay development service that designs synthesizes formulates and delivers analytically quality controlled primer and probe for gene expression assays based on sequence information submitted by the customer For more information contact your Applied Biosystems representative Designing Your Own Al
33. or online web ordering before 10 00 a m PST PDT Orders received by fax will be entered into the order system on the next business day Quantities are based on an average oligo length of 23mer Contact information To order online http store appliedbiosystems com To order by email OligosUS appliedbiosystems com Sequence Detection Systems Chemistry Guide C 13 Appendix C Part Numbers C 14 Sequence Detection Systems Chemistry Guide Index Numerics 3 primer end and amplicon sites 3 11 5 nuclease quantification assays 3 17 5 probe end and amiplicon sites 3 11 A absolute quantification standard curve 3 46 troubleshooting 6 6 absolute standard curve performing 3 46 allelic discrimination assay and TaqMan chemistry 4 2 category 1 2 conclusions 4 6 described 4 2 designing yourown 4 6 guidelines for probes 4 7 how it works 4 3 instruments 4 2 mismatches 4 3 optimizing 4 6 Primer Express software 4 6 reagent configurations 4 8 TaqMan MGB probes 4 6 terms defined 4 2 thermal cycling parameters 4 9 troubleshooting 6 6 amplicon sites and 3 primerend 3 11 and 5 probe end 3 11 and melting temperature 3 11 G C content 3 11 screening 3 10 selection 3 10 amplicons selecting small 3 10 analyzed run data troubleshooting allelic discrimination 6 7 Applied Biosystems contacting xi customer feedback on documentation x Services and Support xi Technical Communications x Technical Support xi Seque
34. pair probe amplifies the endogenous control in the same tube You can perform a multiplex reaction for both the standard curve method and the comparative C4 method The advantages of performing target and reference reactions in the same tube are Higher throughput is most evident if you are interested in analyzing a single target because the number of sample tubes is reduced by a factor of two More efficient use of samples Reduction in reagent use and cost See Primer Limiting in Multiplex Assays on page 3 23 The TaqMan Probe based chemistry includes multiple reporter dyes which make it possible to amplify and detect target amplicon and endogenous control amplicon in the same tube The reporter dyes recommended for TaqMan probes are FAM and VIC These dyes are distinguishable from one another because they have different emission wavelength maxima e FAM Aix 518 nm e VIC max 554 nm The software for all of the SDS instruments uses a process called multicomponenting to distinguish reporter dyes the quencher dye TAMRA A ax 582 nm if used and the passive reference ROX dye Anax 610 nm max max Multicomponenting is a mathematical algorithm that uses pure dye reference spectra to calculate the contribution of each dye to a complex experimental spectrum When using TaqMan MGB probes no quencher dye TAMRA is necessary Because there is one less dye to resolve spectral resolution is improved Ob
35. plates For quantification normalized to an endogenous control standard curves are prepared for both the target and the endogenous control For each experimental sample the amount of target and endogenous control is determined from the appropriate standard curve Then the target amount is divided by the endogenous control amount to obtain a normalized target value Again one of the experimental samples is the calibrator or 1x sample Each of the normalized target values 1s divided by the calibrator normalized target value to generate the relative expression levels e Pipette correctly and accurately Pipette volumes greater than 5 ul of nucleic acid sample to minimize inaccuracy Endogenous Amplification of an endogenous control can be performed to standardize the amount Control of sample RNA or DNA added to a reaction For the quantification of gene expression researchers have used D actin glyceraldehyde 3 phosphate dehydrogenase GAPDH 18S ribosomal RNA rRNA or other RNAs as an endogenous control When using the comparative C4 method with real time PCR you can normalize to an endogenous reference using data generated during the PCR experiment This is especially useful when you have a limited amount of RNA or when performing high throughput processing of a large number of samples Livak and Schmittgen 2001 Another approach is to normalize to a measurement external to the PCR experiment For example you can use UV absor
36. primer concentrations of 900 nM forward and reverse primers The strong amplification of the NTC wells shown in Figure 3 3 a indicates that significant nonspecific amplification is occurring This 1s confirmed by the dissociation curve data shown in Figure 3 3 b which shows that the melting Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay temperature of the product generated in the absence of template is lower than the melting temperature of the specific product generated with template This is typical of primer dimer formation and indicates that lower primer concentrations may provide more optimal results amplification Controls non specific amplification 15 20 2 Cycle Number rget Amplification Derivative Raw Fluorescence Temperature C Figure 3 3 Amplification data using SYBR Green I dye chemistry a Amplification plot linear view demonstrating suspected nonspecific amplification in NTC wells b Dissociation curve analysis confirming product in NTC wells has a different melting temperature from the specific product Optimizing the Probe Concentration The recommended probe concentration of 250 nM ensures excellent assay performance However depending on the requirements of the assay a probe optimization experiment can prove useful Note No probe is required for SYBR Green I dye detection Sequence Detection Systems Chemistry Guide 3 19 Chapter 3
37. probe sequences in allelic discrimination assays Livak et al 1995 Livak et al 1999 Allele Xe 1 Legend d vc Match Mismatch P FAM Q Quencher Allele 2 A AmpliTaq Gold DNA Polymerase Match Mismatch GR1556 Figure 4 1 Results from matches and mismatches between allele and probe sequences in allelic discrimination assays The table below summarizes the possible results of the allelic discrimination assays example shown above A substantial increase in Indicates VIC fluorescence only homozygosity for Allele 1 FAM fluorescence only homozygosity for Allele 2 both fluorescent signals heterozygosity Purchasing an Applied Biosystems Preformulated or Custom Designed Allelic Discrimination Assay Assays on Demand SNP Genotyping Products Product Description 4 4 Assays on Demand SNP Genotyping Products provide the widest collection of biologically informative predesigned quality tested and validated assays for human SNPs that are ready to use on an ABI PRISM Sequence Detection System Assays were designed using Applied Biosystems powerful bioinformatics pipeline and software as well as genomic information from Celera Genomics and public databases Each assay was validated on 90 genomic DNA samples to ensure the highest quality assay and to provide allele frequency information Sequence Detection Systems Chemistry Guide Purchasing an Applie
38. the Applied Biosystems 7900HT Real Time PCR System when using the available automation accessory with 96 well plates C 12 Sequence Detection Systems Chemistry Guide Custom Oligonucleotide Synthesis Custom Oligonucleotide Synthesis Part Number Item Delivery Time 4316034 TaqMan MGB Probe 5 000 to 6 000 pmoles 6 to 7 days 5 Fluorescent label 6 FAM VIC or TET 4316033 TaqMan MGB Probe 15 000 to 25 000 pmoles 6 to 7 days 5 Fluorescent label 6 FAM VIC or TET 4316032 TaqMan MGB Probe 50 000 to 100 000 pmoles 6 to 7 days 5 Fluorescent label 6 FAM VIC or TET 450025 TaqMan TAMRA Probe 5 000 to 6 000 pmoles 4 to 5 days 5 Fluorescent label 6 FAM VIC or TET 450024 TagMan TAMRA Probe 15 000 to 25 000 pmoles 4 to 5 days 5 Fluorescent label 6 FAM VIC or TET 450003 TagMan TAMRA Probe 50 000 to 100 000 pmoles 4 to 5 days 5 Fluorescent label 6 FAM VIC or TET 4304970 Sequence Detection Primers 3 days Minimum 4 000 pmoles purified for sequence detection 4304971 Sequence Detection Primers 4 days Minimum 40 000 pmoles purified for sequence detection 4304972 Sequence Detection Primers 4 days Minimum 130 000 pmoles purified for sequence detection a For Custom Oligonucleotide Synthesis consider the following Delivery includes only business week days orders to be received by email
39. the following Relative Standard Curve Method Running the target and endogenous control amplifications in separate tubes and using the relative standard curve method of analysis requires the least amount of optimization and validation Comparative C Method To use the comparative C4 method a validation experiment must be run to show that the efficiencies of the target and endogenous control amplifications are approximately equal The advantage of using the comparative C method is that the need for a standard curve is eliminated This increases throughput because wells no longer need to be used for the standard curve samples It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples Here the target and endogenous control amplifications are run in separate tubes Multiplex PCR To amplify the target and endogenous control in the same tube limiting primer concentrations must be identified and shown not to affect Cy values By running the two reactions in the same tube throughput is increased and the effects of pipetting errors are reduced What Is Absolute Absolute quantification determines the input copy number of the transcript of Quantification interest usually by relating the PCR signal to a standard curve Livak and Schmittgen 2001 Example Use absolute quantification to measure viral copy number in samples for which this information is not known unknown samples In order to m
40. 384 Well Block Module 4329003 ABI Prism 7900HT Real Time PCR System with 96 Well Block Module 4329007 ABI PRISM 7900HT Real Time PCR System Automation Accessory Upgrade 4331406 ABI PRISM 7900HT Real Time PCR System 384 Well Block Upgrade Kit 4331405 ABI PRISM 7900HT Real Time PCR System 96 Well Block Upgrade Kit 4330087 ABI PRISM 7000 Sequence Detection System 4349042 Applied Biosystems 7300 Real Time PCR system without computer 4349142 Applied Biosystems 7300 Real Time PCR system with laptop computer 4349143 Applied Biosystems 7300 Real Time PCR system with tower computer 4349043 Applied Biosystems 7500 Real Time PCR system without computer 4349144 Applied Biosystems 7500 Real Time PCR system with laptop computer 4349145 Applied Biosystems 7500 Real Time PCR system with tower computer C 2 Sequence Detection Systems Chemistry Guide Sequence Detection Kits Sequence Detection Kits Part Number Kit Instrument 4307266 TaqMan Cytokine Gene Expression Plate 1 with TaqMan Universal ABI PRISM 7700 and PCR Master Mix and Control Total RNA 7000 Sequence Two MicroAmp Optical 96 well Reaction Plates pre loaded with Deteciicn Systems anq Applied Biosystems TaqMan primers and probes for 12 human cytokine targets replicates A 7900HT Real Time PCR of eight and the 18S Ribosomal RNA endogenous control in all System 96 wells TaqMan primer and probe concentrat
41. 40 nM 20 nM Forward 80 nM 80 nM 80 nM 80 nM 80 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 60 nM 60 nM 60 nM 60 nM 60 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 40 nM 40 nM 40 nM 40 nM 40 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 20 nM 20 nM 20 nM 20 nM 20 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Sequence Detection Systems Chemistry Guide 3 23 Chapter 3 Gene Expression and Other Quantitative Assays 3 24 Example The results of a limiting primer matrix experiment are shown in Figure 3 5 Figure 3 5 a shows that only when lowering the primer concentrations below approximately 50 nM is the C value significantly affected Figure 3 5 b shows the corresponding relationship between primer concentrations and AR and demonstrates that lower product yields can be achieved by decreasing forward and reverse primer concentrations The plateau area visible in Figure 3 5 a shows the region in which suitable primer limiting concentrations can be found In this area the C and therefore the corresponding quantification value is unchanged whereas the AR value and corresponding product yield are significantly reduced For this example an appropriate selection of primer limiting concentrations would be at least 50 nM forward and reverse primer It is important to note that probe concentration should be kept at an optimal level even when an assay is primer limited to ensure that the signal produced is large
42. APDH c mycy c myCy ng Total Raji RNA ng Total Raji RNA Norm to GAPDH Rel to Brain Brain 0 033 0 51 Designated calibrator 0 043 0 56 0 036 0 59 0 043 0 53 0 039 0 51 0 040 0 52 Average 0 039 0 004 0 54 0 034 0 07 0 008 1 0 0 12 Kidney 0 40 0 96 0 41 1 06 0 41 1 05 0 39 1 07 0 42 1 06 0 43 0 96 Average 0 41 0 016 1 02 0 052 0 40 0 025 5 5 0 35 Liver 0 67 0 29 0 66 0 28 0 70 0 28 0 76 0 29 0 70 0 26 0 68 0 27 Average 0 70 0 036 0 28 0 013 2 49 0 173 34 2 2 37 Sequence Detection Systems Chemistry Guide 3 33 Chapter 3 Gene Expression and Other Quantitative Assays Table 3 14 Amounts of c myc and GAPDH in human brain kidney liver and lung tissues continued Ti amp s e c myc GAPDH c mycy c myCy ng Total Raji RNA ng Total Raji RNA Norm to GAPDH Rel to Brain Lung 0 97 0 82 0 92 0 88 0 86 0 78 0 89 0 77 0 94 0 79 0 97 0 80 Average 0 93 0 044 0 81 0 041 1 15 0 079 15 7 1 09 a The c myc value is determined by dividing the average c myc value by the average GADPH value The standard deviation of the quotient is calculated from the standard deviations of the c myc and GADPH values b The calculation of c myc relative to brain involves division by the calibrator value This is a division by an arbitrary constant so the cv of this result is the same as the cv for c mycy 3 34 Sequence Detection Systems Chemistry Guide Comparative CT Method for
43. C value from the average c myc C value For example AC Brain 30 49 23 63 6 86 The standard deviation of the difference is calculated from the standard deviations of the c myc and GAPDH values b The calculation of AAC involves subtracting AC calibrator value from the AC target value For example AAC Kidney AC Kidney AC Brain 4 37 6 86 2 50 using full data value not the rounded data presented in this table This is subtraction of an arbitrary constant so the standard deviation of AAC is the same as the standard deviation of the AC value c The range given for c myc relative to brain is determined by evaluating the expression 2 44Cr with AAC s and AAC s where s the standard deviation of the AAC value For example Range Kidney has a AAC value of 2 5 and a range of Standard deviation 0 10 Therefore 2 A4Cr 2 2 50 5 6 The lower range limit 2 50 0 1 5 3 The upper range limit 2 50 0 1 6 0 Sequence Detection Systems Chemistry Guide 3 39 Chapter 3 Gene Expression and Other Quantitative Assays Multiplex PCR Same Tube Method for Relative Quantification Advantages of Multiplex PCR About Multiple Reporter Dyes About Multi componenting About Primer Limitation Multiplex PCR is the use of more than one primer pair and probe in the same tube You can use this method in relative quantification where one primer pair probe amplifies the target and another primer
44. GAPDH mRNA The target and endogenous control are amplified in separate tubes The unknown samples are cDNA prepared from total RNA isolated from human brain kidney liver and lung Sequence Detection Systems Chemistry Guide 3 37 Chapter 3 Gene Expression and Other Quantitative Assays 3 38 Performing the Run The procedure below is a general outline for performing a run Refer to your instrument user manual for detailed instructions To perform the run 1 Set up a reaction plate 2 Place the reaction plate on your instrument and start the run 3 When the run is complete analyze the data Determining the AC Value and Standard Deviation The AC value is determined by subtracting the average GAPDH C value from the average c myc C value The standard deviation of the difference is calculated from the standard deviations of the c myc and GAPDH values using the following formula NE eae S 8 S2 where s std dev As an example from Table 3 16 on page 3 39 brain sample s 0 15 and s 0 09 s A 0 15y 0 09 0 17 Performing the AAC Calculation To perform the AAC calculation 1 Determine the AC by subtracting the average Cy of your endogenous control from the average C4 of your target 2 Determine the AAC by subtracting the AC of your calibrator from the AC of your test sample or treated sample Comparative C Method Results T
45. Gene Expression and Other Quantitative Assays Recommended Probe Concentrations 3 20 The recommended probe concentrations for DNA and cDNA Quantification Assays using TaqMan Probe based chemistry is 250 nM Figure 3 4 shows the results of a probe optimization experiment in which the probe concentration is varied from 50 to 250 nM Figure 3 4 a shows an increase in AR as the probe concentration is increased whereas Figure 3 4 b shows that the C4 value changes for all concentrations of probe It should be noted however that to ensure the best reproducibility especially when wishing to detect low copy numbers of a target sequence it is necessary to avoid probe limiting concentrations The assay should be run at a probe concentration of 250 nM By using a 250 nM concentration probe limitation is avoided and large AR values are ensured Large AR values indicate a robust assay that is performing at high efficiency giving high product yield and allowing more accurate peak measurement Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay a Linear 1 500 250 nM Probe 1300 1 100 150 nM Probe 1000 E1 100 nM Probe ARn 50 nM Probe b Log view 250 nM Probe 150 nM Probe 100 nM Probe 50 nM Probe 1 0001 1 m E 1 000 E2 1 000 E3 Cycle Figure 3 4 Amplification plot linear and log views of probe concentration titration f
46. LD CYCLE 2 min 50 C 10 min 95 C 15 sec 92 C 1 min 60 C a This step is necessary only if you are using Universal Master Mix with UNG Note These conditions are specific for TaqMan Universal PCR Master Mix Sequence Detection Systems Chemistry Guide 4 9 Chapter 4 Allelic Discrimination Assays 4 10 Sequence Detection Systems Chemistry Guide Plus Minus Assays This chapter covers About Plus Minus Assays Using an IPC 1 2 eee Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit Sequence Detection Systems Chemistry Guide Chapter 5 Plus Minus Assays About Plus Minus Assays Using an IPC What Is a Plus Minus Assay What Is an IPC Instruments A Plus Minus Assay is an endpoint assay that indicates the presence or absence of a specific target sequence in a sample The actual amount of target is not determined Example A Plus Minus Assay might be used to determine if the bacteria Salmonella is present in hamburger meat The results will simply show if Salmonella 1s or is not there the amount of the bacteria present will not be determined An internal positive control IPC is used in Plus Minus Assays to monitor the PCR Plus Minus Assays can be accomplished without an IPC however the IPC ensures that a failed PCR is not mistaken for a negative test result The IPC consists of a template and a probe that is added to each well of a reaction plate Applied Biosyste
47. MEER dead Cee aa wea a 4 2 Terms Used in Allelic Discrimination Analysis llle 4 2 How Allelic Discrimination Assays Work 000 ee eee 4 3 Purchasing an Applied Biosystems Preformulated or Custom Designed Allelic Discrimination Assay 1 0 2 2 0 00 cee RIMIS 4 4 Assays on Demand SNP Genotyping Products 00 eee eee 4 4 Product Description diesa a i a o aa a aaa eaa ee 4 4 Product Properties 2 3 2 sore eng ne Mae EAT Kee ede ew ad we a es 4 5 Assay Contents 2 2 conte sie tensgtiessers er peg cals Gti e ely eles a eee a 4 5 Assays by Design Service 00 cece eee 4 6 Designing Your Own Allelic Assay ssaa aaau aeaa 4 6 Design and Optimization Steps 00 000 es 4 6 COMCIUSIONS e 2 a cits ap exons See es ee ede ea eee Gin ooh wee ee Oe es 4 6 Probe Design Using Primer Express Software 0 cee eee eee 4 6 TaqMan MGB Probes 0 000 cece eee eee 4 6 When to Use TaqMan MGB Probes 000 cece eee eee eee 4 7 Allelic Discrimination Probe Design Guidelines 00 0c eee eee 4 7 Primer Design Using Primer Express Software auaa eee eee 4 8 Primer Design Guidelines 0 cece eee 4 8 Selecting the Appropriate Reagent Configuration lere 4 8 Assays Containing TaqMan MGB Probes 00 cee eee eee 4 8 Using the Universal Thermal Cycling Parameters llle 4 9 Thermal Cycling Parameters 0 000 e
48. Products PN 4331183 Assays for genotyping single nucleotide polymorphisms SNPs The products use the 5 nuclease assay for amplifying and detecting specific SNP alleles in purified human genomic DNA samples Each assay allows researchers to genotype individuals for a specific SNP Designed using Applied Biosystems powerful bioinformatics pipeline and software as well as genomic information from Celera Genomics and public databases Each assay was validated on 90 genomic DNA samples to ensure the highest quality assay and to provide allele frequency information Assays on Demand Gene Expression Products PN 4331182 Assays designed for the detection and quantification of specific nucleic acid sequences These products provide researchers with optimized ready to use 5 nuclease assays for human and mouse transcripts Gene expression quantification is performed in a two step RT PCR in which the PCR step is coupled with a 5 fluorgenic nuclease assay For information on available products and specific product uses contact your Applied Biosystems representative or visit the Applied Biosystems web site See How to Obtain Support on page xi Assays by Assays by Design service is an assay development service that designs synthesizes Design Service formulates and delivers analytically quality controlled primer and probe sets for SNP genotyping and gene expression assays based on sequence information submitted by the customer
49. Quantification Assays depends on your experimental design Relative quantification describes the change in expression of the target gene in a test sample relative to a calibrator sample The calibrator sample can be an untreated control or a sample at time zero in a time course study Livak and Schmittgen 2001 Relative quantification provides accurate comparison between the initial level of template in each sample Example You can compare the level of expression of a gene Gene A between a treated and untreated sample for example a drug treatment relative to the expression of a second gene Gene B The resulting data is the expression of Gene A in treated and untreated samples normalized to the expression of Gene B Sequence Detection Systems Chemistry Guide Relative or Absolute Quantification Calculation Relative quantification can be performed with data from all of the SDS instruments Methods for The calculation methods used for relative quantification are Relative ie Relative standard curve method singleplex PCR Quantification d singlep Comparative Cy method AAC singleplex PCR Relative standard curve method multiplex PCR Comparative Cr method multiplex PCR Note Assays on Demand Product and Assays by Design Service products are intended for use in singleplex reactions Determining Which Method to Use All these methods can give equivalent results When determining which method you want to use consider
50. R Master Mix to be used with custom assays 4308321 5 Pack TagMan Exogenous IPC Reagents VIC Dye 1000 Five of PN 4308323 4308320 TaqMan Exogenous Internal Positive Control Reagents VIC Dye 200 with TaqMan Universal PCR Master Mix PN 4308323 and 4304437 4308335 TaqMan Exogenous Internal Positive Control Reagents Protocol VIC Dye 4316831 TaqMan RNase P Detection Reagents Kit 100 20X primer and probe FAM mix and Human Genomic Control DNA 4310982 TaqMan RNase P Instrument Verification Plate One 96 Well Plate One ABI Prism Optical 96 well Reaction Plate pre loaded and sealed with TaqMan primers and probe to detect and quantitate genomic copies of the human RNase P gene 4323306 TagMan RNase P 384 Well Instrument Verification Plate One 384 Well Plate One ABI PRISM Optical 384 well Reaction Plate preloaded and sealed with complete TagMan primers and probe to detect and quantitate genomic copies of the human RNase P gene C 8 Sequence Detection Systems Chemistry Guide Sequence Detection Reagent Components Sequence Detection Reagent Components Part Number Kits Quantity 4304441 TaqMan 1000 RXN Gold with Buffer A Pack 1000 Reactions 1250 Units AmpliTaq Gold DNA Polymerase 10x TaqMan Buffer A 25 mM MgCl Solution 4305822 Sequence Detection Systems Spectral Calibration Kit 4311235 MultiScribe Reverse Trans
51. RTANT The 5700 System cannot perform multiplex same tube reactions so the 5700 System cannot be used for quantification assays using multiplex PCR Terms commonly used in quantification analysis are defined in Table 3 1 Table 3 1 Terms used in quantification analysis Term Definition Amplicon A short segment of DNA amplified during PCR Amplification plot The graphical display of fluorescence signal versus cycle number Baseline A line fit to the initial cycles of PCR in which there is little change in fluorescence signal Threshold cycle C The fractional cycle number at which the fluorescence passes the threshold Calibrator A sample used as the basis for comparative results Endogenous control Internal control gene present in each experimental sample By using an endogenous control as an active reference you can normalize quantification of a messenger RNA mRNA target for differences in the amount of total RNA added to each reaction Intron A non coding segment of gene Sequence Detection Systems Chemistry Guide About Quantitative Assays Table 3 1 Terms used in quantification analysis continued Term Definition No template control NTC A sample that does not contain template It is used to verify amplification quality Nucleic acid target also called target template Nucleotide sequence that you want to detect or quantify Passive r
52. Relative Quantification Comparative C4 Method for Relative Quantification Formula The comparative C4 method is similar to the relative standard curve method except it uses an arithmetic formula rather than a standard curve to achieve the same result for relative quantification IMPORTANT It is possible to eliminate the use of standard curves for relative quantification as long as a validation experiment is performed For more information on performing a validation experiment see below The amount of target normalized to an endogenous control and relative to a calibrator is given by 2 AACT See Appendix A Formulas for a derivation of the formula Performing the Validation Experiment Before using the AAC method for quantification perform a validation experiment like the one below to demonstrate that efficiencies of target and reference are approximately equal To perform the validation experiment 1 Choose your target and endogenous control 2 Perform a dilution series of different input amounts for your target and endogenous control Note When possible serial dilutions should cover 5 to 6 logs 3 Runthe samples on your SDS instrument 4 Analyze your data 5 Calculate the average Cy and AC values for your target and endogenous control see Table 3 15 Sequence Detection Systems Chemistry Guide 3 35 Chapter 3 Gene Expression and Other Quantitative Assays 3 36
53. S Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste Safety Chemical Waste Nnm HAZARDOUS WASTE Refer to Material Safety Data Sheets xvi Hazard and local regulations for handling and disposal Nees CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Ee CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Sequence Detection Systems Chemistry Guide Chemical Waste Safety Chemical Waste To minimize the hazards of chemical waste Safety Guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and l
54. Sequence Detection Systems Applied Biosystems 7900HT Real Time PCR System and 7300 7500 Real Time PCR Systems ABI PRISM 7000 Sequence Detection System and 7700 Sequence Detection System GeneAmp 5700 Sequence Detection System Chemistry Guide Applied KS Biosystems Copyright 2003 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document NOTICE TO PURCHASER PLEASE REFER TO THE ABI PnisM 7900HT Sequence Detection System and SDS Enterprise Database User Guide the ABI PRISM 7000 Sequence Detection System User Guide the ABI PRISM 7700 Sequence Detection System User s Manual the GeneAmp 5700 Sequence Detection System User s Manual the Applied Biosystems 7300 7500 Real Time PCR System Relative Quantitation Getting Started Guide the Applied Biosystems 7300 7500 Real Time PCR System Plus Minus Getting Started Guide the Applied Biosystems 7300 7500 Real Time PCR SystemAllelic Discrimination Getting Started Guide the Applied Biosystems 7300 7500 Real Time PCR System Absolute Quantitati
55. Systems Chemistry Guide Xv Safety and EMC Compliance Information Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose To have a copy of a document sent by fax or e mail select Fax or Email to the left of the document title in the Search Results page then click RETRIEVE DOCUMENTS at the end of the document list After you enter the required information click View Deliver Selected Documents Now Chemical Safety To minimize the hazards of chemicals Guidelines Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xv Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSD
56. X RT Buffer MgCl Solution 4304134 TagMan Reverse Transcriptase Reagents 10 Pack 2000 Ten of PN N808 0234 N808 0236 TagMan EZ RT PCR Core Reagents 200 1000 Units rTth DNA Polymerase 100 Units AmpErase UNG dUTP dATP dCTP dGTP 5X TaqMan EZ Buffer 25 mM Mn OAc p 403028 TaqMan EZ RT PCR Core Reagents 10 Pack 2000 Ten of PN N808 0236 402877 TaqMan EZ RT PCR Kit Protocol 4310179 SYBR Green RT PCR Reagents 200 SYBR Green PCR Master Mix 4309155 TaqMan Reverse Transcriptase Reagents N808 0234 4310251 SYBR Green PCR Master Mix Protocol Combined protocol for SYBR Green PCR Master Mix and SYBR Green RT PCR Reagents 4322171 High Capacity cDNA Archive Kit Random primers Optimized RT Buffer dNTPs and MultiScribe MULV RNase Inhibitor mix for the conversion of up to 10 ug of total RNA in a single 100 uL reaction to single stranded cDNA 4322169 High Capacity cDNA Archive Kit Protocol C 6 Sequence Detection Systems Chemistry Guide Sequence Detection Reaction Kits with Controls Sequence Detection Reaction Kits with Controls Part Number Kit Number of Reactions N808 0230 TaqMan PCR Reagent Kit 200 TaqMan PCR Core Reagents Kit B actin control reagents and protocol N808 0233 TagMan Gold RT PCR Reagents with controls 200 TagMan PCR Core Reagent Kit TaqMan Reverse Transcriptase Reagents TagMan GAPDH Control Reagents and protocol
57. Xo initial number of target molecules Ex efficiency of target amplification n number of cycles The threshold cycle Cy indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold Thus Xp X x 1 E4 87 Ky where Xt threshold number of target molecules Cry threshold cycle for target amplification Kx constant A similar equation for the endogenous control reaction is Ry R X 1 Ep Kp Sequence Detection Systems Chemistry Guide A 1 Appendix A Formulas A 2 where Rr threshold number of reference molecules Ro initial number of reference molecules En efficiency of reference amplification Crp threshold cycle for reference amplification Kp constant Dividing X4 by R4 gives the following expression X iE T Xs REED E r B xdi 8 ISK cadis The exact values of X4 and Ry depend on a number of factors including Reporter dye used in the probe Sequence context effects on the fluorescence properties of the probe Efficiency of probe cleavage Purity of the probe Setting of the fluorescence threshold Therefore the constant K does not have to be equal to 1 Assuming efficiencies of the target and the reference are the same Ex EQ E X Cr x C g X E zK o OR AC Ky E K where Xn X R the normalized amount of target ACy C
58. alert words also appear in user documentation For more information see Words Safety Alert Words on page xiv How to Obtain More Information Related When using this chemistry guide you may find the following documents to be Documentation helpful references ABI PRISM 7900HT Real Time PCR System and SDS Enterprise Database User Guide PN 4317596 ABI PRISM 7000 Sequence Detection System User Guide PN 4348266 ABI PRISM 7700 Sequence Detection System User s Manual PN 904989 GeneAmp 5700 Sequence Detection System User s Manual PN 4304472 RQ Manager Software User Guide PN 4339753 SNP Manager Software User Guide PN 4338776 Primer Express Software Version 2 0 Users Manual PN 4329500 Applied Biosystems 7300 7500 Real Time PCR System Relative Quantitation Getting Started Guide PN 4347824 Applied Biosystems 7300 7500 Real Time PCR System Plus Minus Getting Started Guide PN 4347821 Applied Biosystems 7300 7500 Real Time PCR System Allelic Discrimination Getting Started Guide PN 4347822 Applied Biosystems 7300 7500 Real Time PCR System Absolute Quantitation Getting Started Guide PN 4347825 Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide PN 4347828 Note For additional documentation see How to Obtain Support on page xi Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its Comments user documents You can e mail your comm
59. an c myc mRNA and the endogenous control is human Standard Curve GAPDH mRNA Method The target and endogenous control are amplified in the same tube Dilutions of a cDNA sample prepared from Total Raji RNA are used to construct standard curves for the c myc and the GAPDH amplifications The unknown samples are cDNA prepared from total RNA isolated from human brain kidney liver and lung Performing the Run The same tube and separate tube procedures for the relative standard curve method are identical with the exceptions noted below To perform the run 1 Perform the run per the procedures on page 3 30 Exception When you set up the reaction plate the target amplicon and endogenous control amplicon are in the same tube Exception AC is calculated for each reaction and then the ACys are averaged 2 Determine the relative values per the procedures in Table 3 13 on page 3 31 Results of the Above Run Table 3 17 shows the results of a same tube experiment using the relative standard curve method In this experiment the target c myc normalized to the endogenous control GAPDH was quantified Averages and deviations are calculated from unrounded data not the rounded data presented here Sequence Detection Systems Chemistry Guide 3 41 Chapter 3 Gene Expression and Other Quantitative Assays Table 3 17 Relative quantification using multiplex PCR with the relative standard curve method
60. ation In this example The target is human c myc mRNA and the endogenous control is human GAPDH mRNA The target and endogenous control are amplified in the same tubes The unknown samples are cDNA prepared from total RNA isolated from human brain kidney liver and lung Procedures The same tube and separate tube procedures for the comparative Cy method are identical with the exceptions noted below To perform the run 1 Perform the validation experiment per the procedures on page 3 35 2 Perform the run per the procedures on page 3 38 Exception When you set up the reaction plate the target amplicon and endogenous control amplicon will be in the same tube Exception Because c myc and GAPDH data are being obtained from the same tube calculations are carried out individually for each well before averaging 3 Determine the AC value per the procedures on page 3 38 4 Perform the AAC calculation per the procedures on page 3 38 Sequence Detection Systems Chemistry Guide 3 43 Chapter 3 Gene Expression and Other Quantitative Assays Results of the Run Table 3 18 shows the AAC calculations for the same tube experiment Table 3 18 Relative quantification using multiplex PCR with the comparative C method AC RET c mycy Tissue c myc C4 GAPDH C4 c myc GAPDH AC iba AC
61. bleshooting Table 6 2 Troubleshooting chemistry problems quantification assays continued Observation Possible Cause s Recommended Action Poor amplification of target in multiplex assay Control is too abundant and is outcompeting the target Optimization of the primer and probe concentrations needs to be performed Primer concentrations of the more abundant control need to be limited while concentrations of the target need to be optimized See User Bulletin 5 ABI PRISM 7700 Sequence Detection System SUBJECT Multiplex PCR with TaqMan VIC9 Probes PN 4306236 or refer to Primer Limiting in Multiplex Assays on page 3 23 of this document for procedures on primer limiting Poor amplification of target in multiplex assay continued Probe degradation Mix and aliquot the probe and primers into single use aliquots to prevent degradation by freeze thaw cycles Amplification of the target in the NTCs Contamination of NTCs Verify that this is true amplification by inspecting the multicomponent view for cleavage of the TaqMan probe Repeat the assay using new components for NTC reaction mix Decrease in fluorescence of the passive reference dye Precipitation or degradation in the TaqMan buffers When using the TaqMan PCR Core Reagent kit be sure to mix the tubes well Use TaqMan Universal PCR Master Mix PN 4304437 Be sure to mix thoroughly to produce a homogeneous solution
62. c Plus Minus Chemistry Sena Discrimination using an IPC p Chapter 4 Chapter 5 SYBR Green Dye Yes No No TaqMan probes Yes Yes Yes a Includes one step reverse transcription polymerase chain reaction RT PCR and two step RT PCR for RNA quantification and DNA cDNA quantification Table 2 3 Chemistry considerations for quantification assays using SYBR Green 1 dye or TagMan Probe based chemistry Chemistry Advantage Limitation SYBR Green Reduces cost no probe is needed Provides amplification of any double stranded DNA sequence Yields melting profile of distinct PCR products allows melt curves to be added on the run Increases sensitivity for detecting amplification products relative to product length Can bind to nonspecific double stranded DNA sequences To avoid false positive signals check for non specific product formation using dissociation curve or gel analysis TagMan probe Increases specificity with a probe Provides multiplex capability allows for labelling probes with different distinguishable reporter dyes which allows detection of two distinct sequences in one reaction tube Optimized assays available Allows 5 nuclease assay to be carried out during PCR Provides combined specificity of primers and a probe Requires specific hybridization between probe and target to generate fluorescent signal Sequence Detection Systems Chemistr
63. criptase 100 ml 5000 Units 400 Rxn 10 uL each 40 Rxn 100 uL N808 0119 RNase Inhibitor 2000 Units N808 0260 dNTP Mixture 10 mM 1mL N808 0128 Oligo d T 16 50 uM 0 1 mL N808 0127 Random Hexamers 50 uM 0 1 mL 4307281 Control Total RNA Human 100 uL 50 ng uL N808 0096 AmpErase Uracil N glycosylase UNG 100 uL 1 unit uL 4312660 Control Total DNA Human 2 tubes each 100 uL 10 ng uL 402929 2096 Glycerol Solution Molecular Biology Grade 100 mL Sequence Detection Systems Chemistry Guide C 9 Appendix C Part Numbers Sequence Detection Calibration Kits Part Number Kit Quantity 4305822 Sequence Detection Systems Spectral Calibration Kit 4323977 Sequence Detection Systems 384 Well Spectral Calibration Kit Two 384 Well Plates Two ABI PRISM Optical 384 Well Reaction Plates One preloaded and sealed Background plate and one preloaded and sealed Spectral Calibration plate with eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC 4328639 ABI PRISM 7900HT Sequence Detection Systems 96 Well Spectral Three 96 well Plates Calibration Kit Three ABI PRISM Optical 96 Well Reaction Plates one preloaded and sealed Background plate and two preloaded and sealed Spectral Calibration plates containing eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC9 4328895 ABI Prism 7000 Sequence Detection Systems Spectra
64. d Biosystems Preformulated or Custom Designed Allelic Discrimination Assay Assays on Demand SNP Genotyping Products provide optimized assays for genotyping single nucleotide polymorphisms SNPs The products use the 5 fluorogenic nuclease assay for amplifying and detecting specific SNP alleles in purified genomic DNA samples Each assay allows you to genotype individuals for a specific SNP Visit the Applied Biosystems web site to view the available products Assays on Demand SNP Genotyping Products PN 4331183 See How to Obtain Support on page xi For more information about ordering Assays on Demand SNP Genotyping Products contact your Applied Biosystems representative Product The following are properties of Assays on Demand SNP Genotyping Products Properties All Assays on Demand SNP Genotyping Products are designed and optimized to work with the TaqMan Universal PCR Master Mix No AmpErase UNG using the same universal thermal cycling conditions This facilitates the workflow for all throughput requirements and in any SNP genotyping study Note The assays can be used with TaqMan Universal PCR Master Mix which includes AmpErase UNG The assays require only three components to 20 ng of purified genomic DNA sample 20X SNP Genotyping Assay Mix specific for each polymorphism 2X TaqMan Universal PCR Master Mix No AmpErase UNG The assays require only one amplification step and an endpoint reading to
65. d DNA 2 During the PCR AmpliTaq Gold DNA Polymerase amplifies the target sequence which creates the PCR product or amplicon 3 The SYBR Green I dye then binds to each new copy of double stranded DNA 4 Asthe PCR progresses more amplicon is created Since the SYBR Green I dye binds to all double stranded DNA the result is an increase in fluorescence intensity proportional to the amount of double stranded PCR product produced Figure 2 1 illustrates this process Step 1 Step 2 The SYBR Green I dye within the During PCR AmpliTaq Gold The SYBR Green dye then SYBR Master Mix immediately DNA Polymerase amplifies binds to each new copy of binds with all double stranded each target double stranded DNA DNA present in the sample Figure 2 1 Representation of how the SYBR Green I dye acts on double stranded DNA during one extension phase of PCR 2 2 Sequence Detection Systems Chemistry Guide TaqMan Probe Based Chemistry TaqMan Probe Based Chemistry Development of TaqMan Probe Based Chemistry How TaqMan Sequence Detection Chemistry Works Polymerization PRIMER B pros Q R REPORTER a Q QUENCHER E a wa 5 PRIMER Step 1 A reporter R and a qpencher Q are attached to the 5 and3 ends of a TaqMan probe Initially intercalator dyes were used to measure real time PCR products The primary disadvantage to these type of probes is that they detect accumulation of both specific and nons
66. e Formula seeee RR RR RR RR A 1 Appendix B References Appendix C Part Numbers Sequence Detection Instruments 000 0 cece eee C 2 Sequence Detection Kits liliis C 3 Sequence Detection PCR Reagent Kits lille C 4 Sequence Detection RT PCR Kits 0 00 eee C 6 Sequence Detection Reaction Kits with Controls 00222 eee eee C 7 Sequence Detection Control Reagents 0 cee eee C 8 Sequence Detection Reagent Components 000 cece eee C 9 Sequence Detection Calibration Kits llli C 10 Sequence Detection Disposables lllllee eee C 11 Custom Oligonucleotide Synthesis llle C 13 Index Sequence Detection Systems Chemistry Guide vii viii Sequence Detection Systems Chemistry Guide Preface How to Use This Guide Purpose of This The Applied Biosystems Sequence Detection Systems Chemistry Guide provides an Guide easy to use reference on various techniques and applications including An introduction to sequence detection chemistries Background information design guidelines and general procedures for the following assay types Gene Expression and Quantification Assays Allelic Discrimination Assays Plus Minus Assays Troubleshooting information Audience This guide is intended for users of Applied Biosystems Sequence Detection Systems instruments and chemistries who have a working knowledge of the p
67. e c myc and GAPDH values using the following formula SEEN 2 CV cv tcv where Stddev cv meanvalue Ile As an example from Table 3 14 on page 3 33 brain sample cy 0 004 1 0 039 and _ 0 034 v2 7 0 54 0 004 0 034 SS e ase ae since CV 1o ev X n 0 12 0 07 n Il s 0 008 Comparing Samples with a Calibrator The normalized amount of target c mycy is a unitless number that can be used to compare the relative amount of target in different samples One way to make this comparison is to designate one of the samples as a calibrator In Table 3 14 on page 3 33 brain is designated as the calibrator brain is arbitrarily chosen because it has the lowest expression level of the target Sequence Detection Systems Chemistry Guide Relative Standard Curve Method Results Relative Standard Curve Method for Quantification Each c mycy value in Table 3 14 on page 3 33 is divided by the brain c myc value to give the values in the final column These results indicate the kidney sample contains 5 5 times as much c myc mRNA as the brain sample liver 34 2 times as much and lung 15 7 times as much Note Averages and standard deviations are calculated from unrounded data not the rounded data presented here Table 3 14 Amounts of c myc and GAPDH in human brain kidney liver and lung tissues Tissue c myc G
68. eading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in USA 11 2003 Part Number 4348358 Rev A an Applera business
69. easure the viral copy number in the unknown samples compare the measurement to a standard curve of known viral copy numbers Since the basis for measurement is a standard curve with known quantities of virus the measurement would be absolute Calculation Absolute quantification can be performed with data from all of the SDS instruments Methods for however the absolute quantities of the standards must first be measured by some Absolute independent means Quantification The calculation method used for absolute quantification 1s the standard curve method Sequence Detection Systems Chemistry Guide 3 27 Chapter 3 Gene Expression and Other Quantitative Assays 3 28 Terms Used For More Information The terms in Table 3 13 are used in this discussion of absolute and relative quantification Table 3 13 Terms used in absolute and relative quantification Control Term Definition Standard A sample of known concentration used to construct a standard curve Reference A passive or active signal used to normalize experimental results Endogenous and exogenous controls are examples of active references Active reference means the signal is generated as the result of PCR amplification The active reference has its own set of primers and probe e Endogenous control This is an RNA or DNA that is present in each experimental sample as isolated By using an endogenous control as an active reference you can normalize quantification
70. ection of amplicons in the 50 to 150 basepair range Small amplicons are favored because they promote high efficiency amplification In addition high efficiency assays enable relative quantification to be performed using the comparative Cy method AAC Livak and Schmittgen 2001 This method increases sample throughput by eliminating the need for standard curves when looking at expression levels of a target relative to a reference control For more information on the comparative Cy method see page 3 33 Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay G C Content Whenever possible primers and probes should be selected in a region with a G C content of 30 to 80 Regions with a G C content in excess of this may not denature well during thermal cycling leading to a less efficient reaction In addition G C rich sequences are susceptible to nonspecific interactions that may reduce reaction efficiency and produce nonspecific signal in SYBR Green I dye assays For this same reason primer and probe sequences containing runs of four or more G bases should be avoided Melting Temperature Selecting primers and probes with the recommended melting temperature Tm allows the use of universal thermal cycling parameters Having the probe T be 8 to 10 C higher than that of the primers is recommended 5 End of Probes Primer Express software does not select probes with a G on the 5 end The quenc
71. eference A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or volume A passive reference dye is included in all SDS PCR reagent kits Reporter dye The dye attached to the 5 end of a TaqMan probe The dye provides a signal that is an indicator of specific amplification Normalized reporter Rn The ratio of the fluorescence emission intensity of the reporter dye to the fluorescence emission intensity of the passive reference dye Delta R AR The magnitude of the signal generated by the given set of PCR conditions The AR value is determined by the following formula R baseline Standard A sample of known concentration used to construct a standard curve By running standards of varying concentrations you create a standard curve from which you can extrapolate the quantity of an unknown sample See Relative Standard Curve Method for Quantification on page 3 29 Unknown sample A sample containing an unknown quantity of template that you want to characterize Figure 3 1 shows a representative amplification plot and includes some of the terms defined above Sequence Detection Systems Chemistry Guide Chapter 3 Gene Expression and Other Quantitative Assays Rn Threshold No Template Contr
72. eic acid target also called target template Nucleotide sequence that you want to detect Sequence Detection Systems Chemistry Guide About Allelic Discrimination Assays Table 4 1 Terms used in allelic discrimination analysis continued Term Definition Passive reference A dye that provides an internal reference to which the reporter dye signal is normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or volume A passive reference dye is included in all SDS PCR reagent kits Reporter dye The dye attached to the 5 end of a TaqMan probe Normalized reporter The normalized intensity of the reporter dye Rn R is calculated by dividing the intensity of the reporter dye by the intensity of the passive reference Unknown The sample you want to classify as either homozygote or heterozygote How Allelic In allelic discrimination assays the PCR includes a specific fluorescent dye labeled Discrimination probe for each allele You can use TAMRA or TagMan MGB labelled probes The Assays Work probes contain different fluorescent reporter dyes FAM dye and VIC dye to differentiate the amplification of each allele Each TaqMan MGB probe contains A reporter dye at the 5 end of each probe VIC dye is linked to the 5 end of the Allele 1 probe FAM dye is linked to the 5 end of the
73. enough for accurate multicomponenting by the Sequence Detection System software E 25255 gg 255 26 O 25 265 J 265 27 B 27 275 LJ 275 26 Bl 25255 C 28 5 20 100 00 Reverse primer nM Forward primer nM m 992 Bg 22 04 94 06 C o5 o8 Bo os B 1 12 B 214 Reverse primer n 100 80 60 id Forward primer nM igure 3 5 Results from Limiting Primer Matrix experiment a Shows how C value is affected by variation in forward and reverse primer concentrations Plateau region indicated shows area where C4 value remains constant b Shows reduction in AR Values as primer concentration are decreased Sequence Detection Systems Chemistry Guide Section 3 2 In This Section Section 3 2 Selecting a Data Analysis Approach and Determining Results Selecting a Data Analysis Approach and Determining Results The following topics are covered in this section Section 3 2 Selecting a Data Analysis Approach and Determining Results 3 25 Data Analys18 u Lu sete eh ea te ears d aa a OY Aa ROE OSA LR 3 26 Relative or Absolute Quantification 0 00 c ccc cence nee 3 26 Relative Standard Curve Method for Quantification 000 3 29 Comparative CT Method for Relative Quantification 3 35 Multiplex PCR Same Tube Method for Relative Quantification 3 40 Standard Curve Method for Absolute Quantification 0 00 3 46 Sequence Detection Systems Chemi
74. entration of IPC primers in the PCR reaction In the PCR reaction the IPC is detected using a VIC dye labeled probe and the target template is detected using a FAM dye labeled probe The TaqMan Exogenous IPC Reagents are designed for endpoint plate read assays only Endpoint detection collects fluorescence data after PCR is complete Sequence Detection Systems Chemistry Guide Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit Available Kits Note The part numbers listed in Table 5 2 are for 200 reactions See Appendix C for a list of available kit sizes The reagent configurations listed below are recommended for Plus Minus Assays Table 5 2 Reagent configurations for plus minus assays Reagent Configuration Part Number TaqMan Exogenous Internal Positive Control Reagents with TaqMan Universal PCR Master Mix with VIC dye 4308320 TaqMan Exogenous Internal Positive Control Reagents Note If you are using this kit you will need to purchase one of the core reagents separately as described below TaqMan Universal PCR Master Mix 4304437 TaqMan PCR Core Reagents Kit N808 0228 4308323 Sequence Detection Systems Chemistry Guide 5 5 Chapter 5 Plus Minus Assays 5 6 Sequence Detection Systems Chemistry Guide Troubleshooting This chapter covers Troubleshooting Quantification Assays Troubleshooting Allelic Discrimination Assays Sequence Detection Systems Chemi
75. ents to techpubs appliedbiosystems com Sequence Detection Systems Chemistry Guide How to Obtain Support 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 Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Sequence Detection Systems Chemistry Guide xi Preface xii Sequence Detection Systems Chemistry Guide Safety and EMC Compliance Information This section includes the following topics Safety Conventions Used in This Document 0 2 0 0 eee ee eee xiv General Instrument Safety 0 0 0 cette enn XV Chemical Safety e ea ppi eL Se ELISe REDI Ve USE NEL e DER XV Chemical Waste Safety ace wdc eho ctaa d bursts SEL i D Vae QS xvi Biological Hazard Safety 0 0 cect teens xviii Sequence Detection Systems Chemistry Guide xiii Safety and EMC Compliance Information Safety Conventions Used in This Document xiv Safety Alert Words Four safety aler
76. er extended on MRNA 5 RT om 5 cDNA Tube 1 ue Step Synthesis of 1st cDNA strand 3 Reverse Primer Oligo d T or random hexamer 5 CDNA PCR Step Figure 1 2 Schematic representation of two step RT PCR Table 1 4 summarizes the differences between one and two step RT PCR Comparison of RT PCR Methods Table 1 4 Primers for one and two step RT PCR Method Primers for cDNA Synthesis Features One step Sequence specific reverse primer Requires single reaction mix UNG cannot be used Two step Random hexamers cDNA can be stored for later use Oligo d T 4s UNG can be used Requires two reaction mixes Sequence specific reverse primers An endpoint assay also called a plate read assay measures the amount of accumulated PCR product in fluorescence units at the end of the PCR process The datapoint is the normalized intensity of the reporter dye or R Some endpoint assays can include both pre PCR and post PCR datapoints In this case the system calculates the delta R AR value per the following formula Rn post PCR Rn pre PCR DRn Multiplex PCR is the use of more than one primer probe set in the same tube Multiplex PCR is most commonly used in 5 nuclease quantification assays that involve relative quantification of gene expression Typically one probe is used to detect the target species another probe is used to detect an endogenous control internal control gene
77. ference Sequence Detection Systems Chemistry Guide Chemistry Terms Used in Plus Minus Analysis How Plus Minus Assays Work Sequence Detection Systems Chemistry Guide About Plus Minus Assays Using an IPC Plus Minus Assays using an IPC can be used with fluorogenic 5 nuclease chemistry also known as TaqMan chemistry For information see TaqMan Probe Based Chemistry on page 2 3 Note The SYBR Green I dye chemistry is not supported for Plus Minus Assays using an IPC Terms commonly used in plus minus analysis are defined in the table below Table 5 1 Terms Used in Plus Minus Analysis Term Definition Internal positive control IPC A second TaqMan probe and primer set added to the plate to identify well failure to amplify Provides a means of determining amplification failure that might give a false negative signal NTC No template control NTC A sample that does not contain template The NTC shows background signal and is used as the negative control Provides a means of measuring contamination that might give a false positive signal Nucleic acid target also called target template Nucleotide sequence that you want to detect Unknown sample also called sample of interest The sample for which you want to determine the presence or absence of a specific target Plus Minus Assays begin by aliquoting the following to each well of a plate PCR master mix pr
78. h 4 9 Plus Minus Assays About Plus Minus Assays Using an IPC 00 ee ee 5 2 What Is a Plus Minus Assay sseeeeee tee 5 2 What ls an IPC ck Em 5 2 Instruments vree a a cases Saad ees seas ee Sk b dala NER ES ra p RERO 5 2 GHEMISIIY iunc pEEETAU eae ee wea Oe ee ee a ee ee I NER 5 3 Terms Used in Plus Minus Analysis lees 5 3 How Plus Minus Assays Work 0000s 5 3 Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit 5 4 Kit Features os orc Lo ye ad sees Be qu ae te Saas BRE 5 4 Amplifying the IPC and Target in the Same Tube 00 0 eee eee 5 4 Endpoint Detection and Post PCR Plate Read 0202 cece eeeee 5 4 Available Kits enden eck ae e mese mp3 eee ae E a ota eee 5 5 Sequence Detection Systems Chemistry Guide Chapter 6 Troubleshooting Troubleshooting Quantification Assays 1 6 0 ee ee 6 2 Using SDS Software to Diagnose Irregular Data 00000 eee 6 2 Troubleshooting Chemistry Problems 0002 cece eee eee 6 3 Troubleshooting Allelic Discrimination Assays sellers 6 6 Troubleshooting Run Data seitas samia eee re 6 6 Troubleshooting Analyzed Run Data 0 0c es 6 7 Appendix A Formulas Comparative CT Method for Relative Quantification 0 000 c eens A 1 Formulas ruo stays teli ewes x othe De E ek endl ee TES eases ens p eot esr su ue de Hels d a dante A 1 Derivation of th
79. he C data used to determine the amounts of c myc and GAPDH mRNA shown in Table 3 15 on page 3 37 are used to illustrate the AAC calculation Table 3 16 below shows the average Cy results for the human brain kidney liver and lung samples and how these Cz s are manipulated to determine AC AAC and the relative amount of c myc mRNA The results are comparable to the relative c myc levels determined using the standard curve method Sequence Detection Systems Chemistry Guide Comparative CT Method for Relative Quantification Although the comparative C4 method can be used to make this type of tissue comparison biological interpretation of the results is complex The single relative quantity reported actually reflects variation in both target and reference transcripts across a variety of cell types that might be present in any particular tissue Livak and Schmittgen 2001 Table 3 16 Relative quantification using the comparative C method Endogenous Tissue bass Control AC AAC c mycy A cod GAPDH c myc GAPDH AC AC Brain Rel to Brain erage T Average C Brain 30 49 0 15 23 63x0 09 6 86x0 17 0 00 0 17 1 0 Calibrator 0 9 to 1 1 Kidney 27 03 0 06 22 66 0 08 4 37 0 10 2 50 0 10 5 6 5 3 to 6 0 Liver 26 25 0 07 24 60 0 07 1 65 0 10 5 21 0 10 37 0 34 5 to 39 7 Lung 25 83 0 07 23 01 0 07 2 81 0 10 4 05 0 10 16 5 15 4 to 17 7 a The AC value is determined by subtracting the average GAPDH
80. he input amount by entering the following formula in an adjacent cell 10 cell containing log input amount Note The units of the calculated amount are the same as the units used to construct the standard curve which are nanograms of Total Raji RNA If it is calculated that an unknown has 0 23 ng of Total Raji RNA then the sample contains the same amount of c myc mRNA found in 0 23 ng of the Raji Control RNA Repeat the steps to construct a standard curve for the endogenous reference using the Cr values determined with the GAPDH probe Refer to Table 3 14 on page 3 33 Because c myc and GAPDH are amplified in separate tubes average the c myc and GAPDH values separately Divide the amount of c myc by the amount of GAPDH to determine the normalized amount of c myc c myc Designate the calibrator In Table 3 14 on page 3 33 brain is arbitrarily designated as the calibrator Divide the averaged sample kidney liver or lung value by the averaged calibrator brain value Calculate the coefficient of variation see below based on the cv of the sample and brain Sequence Detection Systems Chemistry Guide 3 31 Chapter 3 Gene Expression and Other Quantitative Assays 3 32 Calculating the Coefficient Of Variation The c myc value is determined by dividing the average c myc value by the average GAPDH value The standard deviation of the quotient is calculated from the standard deviations of th
81. hing effect of a G base in this position will be present even after probe cleavage This can result in reduced fluorescence values AR see Table 3 1 on page 3 4 which can impact the performance of an assay Having G bases in positions close to the 5 end but not on it has not been shown to compromise assay performance 3 End of Primers The last five bases on the 3 end of the primers should contain no more than two C and or G bases which is another factor that reduces the possibility of nonspecific product formation Under certain circumstances such as a G C rich template sequence this recommendation may have to be relaxed to keep the amplicon under 150 basepairs in length In general avoid primer 3 ends extremely rich in G and or C bases Sequence Detection Systems Chemistry Guide 3 11 Chapter 3 Gene Expression and Other Quantitative Assays Summary of Primer and MGB 3 12 Probe Design Guidelines Table 3 3 summarizes primer and probe guidelines for MGB probe design Table 3 3 Primer and probe design guidelines for quantitative assays Probe Guidelines Primer Guidelines Select the probe first and design the primers as close as possible to the probe without overlapping it amplicons of 50 to 150 basepairs are strongly recommended Keep the G C content in the 30 to 80 range Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be av
82. ible in from each well Outlying amplification When the run data is viewed in the C vs Well Position plot do replicate wells amplify comparably Wells producing C values that differ significantly from the average for the associated replicate wells may be considered outliers If a plate produces nonuniformity between replicates some samples on the plate could have evaporated Check the seal of the optical adhesive cover for leaks Troubleshooting Chemistry Problems Chemistry problems are often the cause of abnormal results For example a curve shaped like an upside down U indicates that too much template is present Table 6 2 describes several chemistry problems that may be encountered when running Quantification Assays Table 6 2 Troubleshooting chemistry problems quantification assays Observation Possible Cause s Recommended Action Poor amplification of target Poor quality template Check each template preparation by agarose electrophoresis to determine its purity ensuring that only one product is formed Poor RT conversion to cDNA e Check the RNA sample for degradation Input RNA could be too concentrated or too dilute Make sure serial dilutions of template RNA from original stock and repeat RT PCR Ensure RT PCR setup has been performed under the appropriate conditions to avoid premature cDNA synthesis Sequence Detection Systems Chemistry Guide 6 3 Chapter 6 Trou
83. imers and fluorogenic probes constructed for the target nucleic acid sequence Test samples are then added to the plate and loaded into a thermal cycler for thermal cycling During the PCR the fluorogenic probes anneal specifically to the complementary target sequence between the forward and reverse primer sites on the template DNA Then during extension AmpliTaq Gold DNA polymerase cleaves the hybridized probes in each sample containing the target The cleavage of each matched probe separates the reporter dye from the quencher dye which results in increased fluorescence by the reporter After thermal cycling the plate is run on an SDS instrument which reads the fluorescence generated during the PCR amplification The fluorescent signals measured by the SDS software can determine the presence or absence of the target nucleic acid in each sample on the plate Incorporating an IPC An IPC is a second TaqMan probe and primer set added to the plate that targets a low copy constitutive nucleic acid If a well does not exhibit amplification the SDS software uses the positive signal from the IPC to confirm that the well failed to amplify because of a lack of template rather than a pipetting error 5 8 Chapter 5 Plus Minus Assays The configuration of samples on a plus minus assay plate is slightly different when using an IPC For plates containing an IPC primers probe and template are pipetted with the target assay to all unknown wel
84. ing an Assay Type s kieser bema a a A e a aa a a eee 1 2 SDS Instrument eaii nre a a o E REKA E eee e a oe dace x 1 2 Assay Types Supported 000 ee 1 2 About Real Time PCR Assays 000 e eect teas 1 3 About Endpoint Assays rere ex ea Yee et ee eee E 1 5 About Multiplex PCR 0000 ee 1 5 Selecting the Sequence Detection Chemistry 0 000 eee eee 1 6 TaqMan Probe Based Chemistry llle 1 6 SYBR Green Dye Chemistry lllieeeeleeeeeeeeeee 1 6 Selecting an Assay Source llelleeleeeeee eh 1 7 Assays on Demand Products 000 cece ele 1 7 Assays by Design Service 0 000 c eee 1 7 Designing Your Own Assay z reids uea ay nuaa a A SA ERA EEE n 1 8 Performing the ASSaV si sepespee pte eR EEIE EEEO O Pe Ee Oe 1 9 Selecting a Data Analysis Approach and Determining Results 1 9 Chapter 2 Chemistry Overview SYBR Green Dye Chemistry lseeeeeeeee nh 2 2 Development of SYBR Green Dye Chemistry nuuanu eee eee 2 2 How the SYBR Green Dye Chemistry Works 0 2 000 ellen 2 2 Sequence Detection Systems Chemistry Guide iii Chapter 3 TaqMan Probe Based Chemistry 0 00 eee eee eee 2 3 Development of TaqMan Probe Based Chemistry 2 20000 eee 2 3 How TaqMan Sequence Detection Chemistry Works 22000000 2 3 Two Types of TaqMan Probes 0 00 eee 2 4 Selecti
85. ion about xi troubleshooting allelic discrimination assays 6 6 allelic discrimination run data 6 7 chemistry problems 6 3 DNA concentration effects 6 6 quantification assays 6 2 two step RT PCR Assays on Demand 3 7 primers used 3 6 U UNG and minimizing carryover 2 6 Universal Master Mix reagents about SYBR Green I Mix 3 14 about TaqMan mix 3 14 polymerase benefit 3 14 user attention words defined ix Sequence Detection Systems Chemistry Guide V validation quantification 3 35 W WARNING description xiv waste disposal guidelines xvii Z components sequence detection reagents C 9 custom oligonucleotide synthesis C 13 kits sequence detection calibration C 10 reagents sequence detection control C 8 Sequence Detection Systems Chemistry Guide Index 5 Index 6 Sequence Detection Systems Chemistry Guide 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 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Bipsystems Applera Corporation is committed to providing the world s l
86. ions are optimized for y multiplex PCR utilizing FAM and VIC dyes Configuration includes TaqMan Universal PCR Master Mix MicroAmp Optical Caps Control Total RNA Human and Protocol 4307265 TaqMan Cytokine Gene Expression Plate 1 with TaqMan Universal ABI PRISM 7700 and PCR Master Mix 7000 Sequence Two MicroAmp Optical 96 well Reaction Plates pre loaded with Detection systems ane Applied Biosystems TagMan primers and probes for 12 human cytokine targets replicates 7900HT Real Time PCR of eight and the 18S Ribosomal RNA endogenous control in all System 96 wells TaqMan primer and probe concentrations are optimized for y multiplex PCR utilizing FAM and VIC dyes Configuration includes TaqMan Universal PCR Master Mix and MicroAmp Optical Caps 4306744 TagMan Cytokine Gene Expression Plate 1 Protocol 4309920 TagMan Human Endogenous Control Plate with TaqMan Universal ABI Prism 7700 and PCR Master Mix and Control Total RNA 7000 Sequence Two MicroAmp Optical 96 well Reaction Plates pre loaded with DES OM dx TaqMan primers and probes for 11 human endogenous control targets pp S 2 7900HT Real Time PCR and an internal positive control IPC in replicates of eight TaqMan System primer and probe concentrations are optimized for 50ul reactions and y utilize VIC dye Configuration includes TaqMan Universal PCR Master Mix MicroAmp Optical Caps Control Total RNA Human and Protocol 4309921 TagMan Huma
87. ive Reference 1 and optimized buffer components One 5 mL vial in each box 4324020 TagMan Universal PCR Master Mix No AmpErase UNG 10 Pack 2000 Ten 5 mL vials in each box 4326614 TagMan Universal PCR Master Mix No AmpErase UNG 50 ml 2000 4304449 TaqMan Universal PCR Master Mix Protocol N808 0228 TaqMan PCR Core Reagents Kit 200 250 Units AmpliTaq Gold DNA Polymerase 100 Units AmpEase UNG dUTP dATP dCTP dGTP 10X TaqMan Buffer A 25 mM MgCl Solution 4304439 TaqMan 1000 RXN PCR Core Reagents 1000 1250 Units AmpliTaq Gold DNA Polymerase 500 Units AmpEase UNG dUTP dATP dCTP dGTP 10X TaqMan Buffer A 25 mM MgCl Solution 402930 TaqMan PCR Core Reagent Kit 10 Pack 2000 Ten of PN N808 0228 402823 TaqMan PCR Reagent Kit Protocol 4304886 SYBR Green PCR Core Reagents 200 250 Units AmpliTaq Gold DNA Polymerase 100 Units AmpEase UNG dNTP Mix with dUTP 10X SYBR Green PCR Buffer 25 mM MgCl Solution 4306736 10 Pack SYBR Green PCR Core Reagents 2000 Ten of PN 4304886 C 4 Sequence Detection Systems Chemistry Guide Sequence Detection PCR Reagent Kits 4304965 SYBR Green PCR Core Reagents Protocol 4309155 SYBR Green PCR Master Mix Supplied at a 2X concentration The mix is optimized for SYBR Green reactions and contains SYBR Green 1 dye AmpliTag Gold DNA Polymerase dNTPs with dUTP Passive Reference 1 and optim
88. ization concentration that will give you the minimum C and maximum AR Matrix A primer optimization matrix can help to compensate for nonspecific primer binding which can reduce the amount of primer available to bind at its specific site 5 Nuclease Fora 5 nuclease quantification assay optimal performance is achieved by selecting Quantification the primer concentrations that provide the lowest Cz and highest AR for a fixed Assays amount of target template The results of a typical TaqMan primer optimization matrix experiment are shown in Figure 3 2 Figure 3 2 a shows the amplification plots for all primer concentration combinations in linear view Figure 3 2 b shows the same data in log view format The combination of 50 nM forward and reverse primer Plot group C gives both the lowest AR and highest Cy All other primer combinations that contain a 150 nM concentration of either the forward or reverse primer Plot group B give a reduced AR All primer combinations that contain at least 300 nM forward and reverse primer Plot group A give both the highest AR and the lowest C as a result any of the plot group A or B would provide optimal performance It should be noted that although Cy values are the parameter by which quantitative values are assigned in a real time Quantification Assay AR values can also prove important when trying to obtain maximum sensitivity and reproducibility Sequence Detection Systems Chemistry Guide
89. ized buffer components One 5 mL vial in each box 200 4312704 SYBR Green PCR Master Mix 10 Pack Ten of PN 4309155 2000 4334973 SYBR Green PCR Master Mix 2000 4310251 SYBR Green PCR Master Mix Protocol Combined protocol for SYBR Green PCR Master Mix and SYBR Green RT PCR Reagents Sequence Detection Systems Chemistry Guide C 5 Appendix C Part Numbers Sequence Detection RT PCR Kits Number of 50 uL Part Number Kit Reactions 4309169 TaqMan One Step RT PCR Master Mix Reagents Kit 200 Vial1 AmpliTag Gold DNA Polymerase mix 2X is optimized for TaqMan reactions and contains AmpliTaq Gold DNA Polymerase dNTPs with dUTP Passive Reference 1 and optimized buffer components Vial 2 RT enzyme mix 40X contains MultiScribe Reverse Transcriptase and RNase Inhibitor 4313803 TaqMan One Step RT PCR Master Mix Reagents Kit 10 Pack 2000 Ten of PN 4309169 4310299 TaqMan One Step RT PCR Master Mix Reagents Kit Protocol N808 0232 TagMan Gold RT PCR Reagents without controls 200 TaqMan PCR Core Reagents Kit N808 0228 TaqMan Reverse Transcriptase Reagents N808 0234 4304133 TaqMan Gold RT PCR Reagents without controls 10 Pack 2000 Ten of PN N808 0232 402876 TaqMan Gold RT PCR Protocol N808 0234 TagMan Reverse Transcriptase Reagents 200 MultiScribe Reverse Tanscriptase RNase Inhibitor dNTP Mixture Oligo d T s Random Hexamers 10
90. kg 4326659 ABI PRISM 96 Well Optical Reaction Plate with Barcode code 128 500 Plates Pkg 25 Pack 25X PN 4306737 ABI PRISM 96 Well Optical Reaction Plate with Barcode code 128 N801 0560 MicroAmp Optical 96 Well Reaction Plate 10 Plates Pkg Not Barcoded 403012 MicroAmp Optical 96 Well Reaction Plates and ABI PRISM Optical 20 Plates Caps PN 4306737 and 4323032 2400 Caps Pkg 4323032 ABI PRISM Optical Caps 8 Caps Strip 300 Strips Pkg Flat Caps 2400 Caps Pkg 4312063 MicroAmp Splash Free Support Base for 96 Well Reaction Plates 10 Bases Pkg 4316567 ABI PRISM Optical Tubes 8 Tubes Strip 125 Strips Pkg For use on ABI PRISM 7000 7700 and GeneAmp 5700 Sequence 1000 Tubes Pkg Detection Systems only 403081 MicroAmp 96 Well Tray Retainer Sets 10 Sets For use with single or strip optical tubes on the ABI PRISM 7000 7700 and GeneAmp 5700 Sequence Detection Systems Sequence Detection Systems Chemistry Guide C 11 Appendix C Part Numbers N801 0933 MicroAmp Optical Tubes 2000 Pkg For use on ABI Prism 7000 7700 and GeneAmp 5700 Sequence Detection Systems only 4330015 ABI PRISM Cap Installing Tool One Pkg Used for removing MicroAmp Caps or ABI PRISM Optical Caps from the MicroAmp plate or ABI PRISM 96 Well Optical plate and Tray Retainer Assemblies 433292 ABI PRISM Snap On Compression Pads 9 Pkg Snap On Compression Pad is a compliant cover bonded to a metal frame and is designed for use on
91. l Calibration Kit Eight 96 Well Plates Eight ABI PRISM Optical 96 Well Reaction Plates one preloaded and sealed Background plate and seven preloaded and sealed Spectral Calibration plates containing seven separate dye standards FAM JOE NED ROX SYBR Green TAMRA VIC C 10 Sequence Detection Systems Chemistry Guide Sequence Detection Disposables Sequence Detection Disposables Part Number Item Quantity 4313663 ABI PRISM Optical Adhesive Cover Starter Kit 20 Covers Pkg ABI PRISM Optical Adhesive Covers quantity 20 Applicator quantity 1 ABI PRISM Optical Cover Compression Pad quantity 1 4311971 ABI PRISM Optical Adhesive Covers 100 Covers Pkg 4314320 ABI PRISM Optical Adhesive Covers and ABI PRISM 96 Well Optical 100 Covers Reaction Plate with Barcode code 128 100 Plates Pkg PN 4311971 and 5X ABI PRISM Optical 96 Well Reaction Plates PN 4306737 4312639 ABI PRISM Optical Cover Compression Pads 5 Pads Pkg 4333183 Adhesive Seal Applicators 5 Pkg 4309849 ABI PRisM 384 Well Clear Optical Reaction Plate with Barcode 50 Plates Pkg code 128 4326270 ABI PRISM 384 Well Clear Optical Reaction Plate with Barcode code 500 Plates Pkg 128 10 Pack 10X PN 4309849 ABI PRISM 384 Well Clear Optical Reaction Plate with Barcode code 128 4306737 ABI PRISM 96 Well Optical Reaction Plate with Barcode code 128 20 Plates P
92. lative Abundance of the Target and Reference In applying the primer limitation to target and endogenous control amplifications the relative abundance of the two species must be considered For quantification of gene expression it is possible to use rRNA as an endogenous control The concentration of rRNA in total RNA is always greater than the concentration of any target mRNA Therefore in multiplex reactions amplifying both target and rRNA only the concentrations of the rRNA primers need to be limited Limiting Primer Matrix To define limiting primer concentrations run a matrix of forward and reverse primer concentrations using the value of the minimum initial template The goal is to identify primer concentrations that reduce the AR of the assay without affecting the Cy value Table 3 12 illustrates a recommended matrix of forward and reverse primers varying in concentration from 20 to 100 nM Note Although following all design criteria does facilitate the ability to identify limiting primer concentrations it may not be possible for all assays If a limiting primer matrix experiment does not enable the identification of primer limiting concentrations it will be necessary to redesign at least one primer or run the reactions in separate tubes Table 3 12 Matrix of varying concentrations of forward and reverse primers 20 to 100 nM Forward 100 nM 100 nM 100 nM 100 nM 100 nM Reverse 100 nM 80 nM 60 nM
93. le 2 Allelic discrimination assays can be used with the following Sequence Detection System SDS instruments Applied Biosystems 7900HT Real Time PCR System 7900HT System Applied Biosystems 7300 Real Time PCR System 7300 System Applied Biosystems 7500 Real Time PCR System 7500 System ABI PRISM 7000 Sequence Detection System 7000 System ABI PRISM 7700 Sequence Detection System 7700 System Note Use of the 7900HT system 7000 system or 7700 system allows for real time analysis of PCR which is helpful for troubleshooting If using a sequence detection system for PCR amplification perform the endpoint plate read separately Note The GeneAmp 5700 Sequence Detection System 5700 System cannot be used for allelic discrimination assays Allelic Discrimination Assays can be used with fluorogenic 5 nuclease chemistry also known as TaqMan Probe based chemistry For information see TaqMan Probe Based Chemistry on page 2 3 Note The SYBR Green I dye chemistry is not supported for Allelic Discrimination Assays Terms commonly used in allelic discrimination analysis are defined in Table 4 1 Table 4 1 Terms used in allelic discrimination analysis Term Definition No template control NTC A sample that does not contain template The NTC shows background signal and is used as the negative control Provides a means of measuring contamination that might give a false positive signal Nucl
94. lelic Assay Design and Optimization Steps Conclusions This section discusses the Applied Biosystems Assay Design Guidelines developed specifically for allelic discrimination assays Use these guidelines only when designing your own assays These guidelines contain the following important steps Designing probes and primers using Primer Express software Selecting the appropriate reagent configuration Using universal thermal cycling parameters Using default primer and probe concentrations IMPORTANT These steps provide a rapid and reliable system for assay design and optimization only when used in their entirety In order to achieve the highest level of success use the system as a whole Many of the individual components are interdependent The Applied Biosystems Assay Design Guidelines enable allelic discrimination assays to be designed and optimized rapidly and efficiently Since thousands of assays have been developed this way the following conclusion can be made To attain a highly reproducible and sensitive assay you can use 900 nM primers a 200 nM probe and 1 to 20 ng of genomic DNA Probe Design Using Primer Express Software TaqMan MGB Probes The Primer Express software uses a set of default parameters to automatically select primer and probe sets The probe design guidelines for allelic discrimination assays are discussed here After choosing probes based on the guidelines below you design the primer
95. ls on the plate In addition to the NTC and unknown samples a positive control for the IPC is also arrayed on the plate This group of wells IPC contains IPC template IPC primers and probe target primers and probe but no target template Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit Kit Features Amplifying the IPC and Target in the Same Tube Endpoint Detection and Post PCR Plate Read The Applied Biosystems TaqMan Exogenous Internal Positive Control Reagents kit contains a pre optimized internal positive control IPC that can be spiked into samples to distinguish true target negatives from PCR failure due to inhibition The kit is designed to Distinguish types of negative results A negative call for the target sequence and positive call for the IPC indicates that no target sequence is present A negative call for the target sequence and negative call for the IPC suggests PCR inhibition Avoid amplification of endogenous genes Permit co amplification of the IPC and the target sequence without compromising amplification of the target sequence Perform optimally with the TaqMan Universal PCR Master Mix By using the TaqMan Exogenous IPC Reagents a low copy target DNA can be amplified in the same tube with the IPC Although the target and IPC DNAs may differ in initial copy number the amplification efficiency of the target reaction 1s not compromised This is achieved by limiting the conc
96. method comparison 1 5 one step 1 4 two step 1 4 run outline for performing 3 30 S safety before operating the instrument xv biological hazards xviii chemical xv chemical waste xvi conventions xiv guidelines xvi xvii moving lif ng xv sample comparisons using a calibrator 3 32 Services and Support obtaining xi small amplicons selecting 3 10 SNP genotyping assay contents 4 5 assay information 4 5 assay mix 4 5 Assays by Design 4 6 Assays on Demand 4 4 properties of assays 4 5 standard curve absolute quantification 3 46 requriements for quantification 3 29 standard deviation determining 3 38 standardization and use of endogenous control 3 29 standards absolute quantities 3 46 requirements for preparation 3 29 SYBR Green I dye chemistry described 1 6 optimizing quantification assays 3 18 Index 4 T TAMRA dye and TaqMan probe 2 4 TaqMan chemistry described 1 6 2 3 plus minus assays 5 3 specific detection 2 3 used by 1 6 TaqMan exogenous IPC endpoint assays 5 4 kit design 5 4 TaqMan MGB probes 2 4 allelic discrimination assay 4 6 usage 2 4 when to use 4 7 TaqMan probes types 2 4 target assays and Assays on Demand 3 8 Technical Communications contacting x e mail address x Technical Support contacting xi thermal cycling parameters allelic discrimination assays 4 9 DNA cDNA quantification 3 15 recommendations 3 15 RNA one step 3 15 RNA quantification 3 15 RNA two step 3 16 training obtaining informat
97. ms has developed the TaqMan Exogenous Internal Positive Control Reagents kit for use in Plus Minus Assays These reagents in conjunction with your target identify samples that are positive or negative for a specific target sequence The kit distinguishes between two types of negative reactions Samples identified as negative because they lack the target sequence Samples identified as negative because of the presence of a PCR inhibitor Note For more detailed information see Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit on page 5 4 Plus Minus Assays using an IPC can be used with the following Sequence Detection System SDS instruments ABI PnisM 7000 Real Time PCR System 7000 System Applied Biosystems 7300 Real Time PCR System 7300 System Applied Biosystems 7500 Real Time PCR System 7500 System ABI PRISM 7700 Sequence Detection System 7700 System Applied Biosystems 7900HT Real Time PCR System 7900 System Note The GeneAmp 5700 Sequence Detection System 5700 System cannot be used for Plus Minus Assays using an IPC These instruments are used to measure the increase of reporter fluorescence following PCR Reporter signals are normalized to the emission of a passive reference as follows Raq Emission Intensity of Target Template Sequence Emission Intensity of Passive Reference Rn pc Emission Intensity of Internal Positive Control Emission Intensity of Passive Re
98. n 1 10 Sequence Detection Systems Chemistry Guide Chemistry Overview This chapter covers SYBR Green I Dye Chemistry TaqMan Probe Based Chemistry Selecting the Appropriate Chemistry Minimizing DNA Contaminants Sequence Detection Systems Chemistry Guide 2 1 Chapter 2 Chemistry Overview SYBR Green Dye Chemistry Development of Small molecules that bind to double stranded DNA can be divided into two classes SYBR Green intercalators and minor groove binders MGBs Higuchi Higuchi et al 1992 used Dye Chemistry the intercalator ethidium bromide for their real time detection of PCR Hoechst 33258 is an example of a minor groove binding dye whose fluorescence increases when bound to double stranded DNA Higuchi et al 1993 Regardless of the binding method there are at least two requirements for a DNA binding dye for real time detection of PCR products Increased fluorescence when bound to double stranded DNA No inhibition of PCR Applied Biosystems has developed conditions that permit the use of the SYBR Green I dye in PCR without PCR inhibition and with increased sensitivity of detection compared to ethidium bromide How the SYBR The SYBR Green I dye chemistry uses the SYBR Green I dye to detect PCR Green Dye products by binding to double stranded DNA formed during PCR Here s how it Chemistry Works works 1 When SYBR Master Mix is added to a sample SYBR Green I dye immediately binds to all double strande
99. n Endogenous Control Plate with TagMan Universal ABI PRISM 7700 and PCR Master Mix 7000 Sequence Two MicroAmp Optical 96 well Reaction Plates pre loaded with pelo d ae TaqMan primers and probes for 11 human endogenous control targets pp yS 7900HT Real Time PCR and an internal positive control IPC in replicates of eight TaqMan System primer and probe concentrations are optimized for 50ul reactions and y utilize VIC dye Configuration includes TaqMan Universal PCR Master Mix and MicroAmp Optical Caps 4308134 TagMan Human Endogenous Control Plate Protocol Sequence Detection Systems Chemistry Guide C 3 Appendix C Part Numbers Sequence Detection PCR Reagent Kits Number of 50 uL Part Number Kit 2 Reactions 4304437 TaqMan Universal PCR Master Mix 200 Supplied at a 2X concentration The mix is optimized for TaqMan reactions and contains AmpliTaq Gold DNA Polymerase AmpErase UNG dNTPs with dUTP Passive Reference 1 and optimized buffer components One 5 mL vial in each box 4318157 TaqMan Universal PCR Master Mix 2000 Ten 5 mL vials in each box 4305719 TaqMan Universal PCR Master Mix 10 Pack 2000 Ten boxes of PN 4304437 4326708 50 ml TagMan Universal PCR Master Mix 2000 4324018 TagMan Universal PCR Master Mix No AmpErase UNG 200 Supplied at a 2X concentration The mix is optimized for TaqMan reactions and contains AmpliTaq Gold DNA Polymerase dNTPs with dUTP Pass
100. nce Detection Systems Chemistry Guide assay design considerations 3 9 guidelines 1 8 options 1 7 Primer Express software 3 9 assay development service Assays by Design 3 8 assay source selecting 1 7 assay type selection of 1 2 supported types 1 2 using SYBR Green I chemistry 1 6 using TaqMan chemistry 1 6 Assays by Design about 1 7 and special needs 3 8 SNP genotyping 4 6 Assays on Demand product types 1 7 properties 3 8 purpose 3 7 SNP genotyping 4 4 B biohazardous waste handling xvii biological hazard guidelines xviii bold text when to use ix C calculation methods absolute quantification 3 27 calibrator for sample comparisons 3 32 carryover UNG to minimize 2 6 CAUTION description xiv chemicalsafety xv xvi chemical waste safety xvi xvii chemistries selecting 1 6 chemistry choice assay type 2 5 quantification assays 2 5 selection criteria 2 5 coefficient of variation calculating 3 32 Index 1 comparative CT example 3 37 formula 3 35 how used 3 38 multiplex PCR 3 43 performing 3 37 relative quantification 3 35 contamination minimizing DNA 2 6 conventions bold text ix IMPORTANTS ix in this guide ix italic text ix menu commands ix Notes ix safety xiv user attention words ix CT calculation relative efficiency 3 37 customer feedback on Applied Biosystems documents x cv calculating 3 32 D DANGER description xiv data analysis gene expression 3 26
101. ned and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles rene CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles 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 receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems This service is free and available 24 hours a day To obtain MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 In the Search field type in the chemical name part number or other information that appears in the MSDS of interest Select the language of your choice then click Search Sequence Detection
102. ng the Appropriate Chemistry 000 0c eee eee eee 2 5 Minimizing DNA Contaminants 0 0 0 tee 2 6 Using UNG to Minimize Reamplification Carryover Products 2 6 General PCR Practices cricar rore rito teina eee nas 2 7 Gene Expression and Other Quantitative Assays Section 3 1 Introduction 0 ER e A A EE eee eee 3 3 In ThiS Sectiori espe Pee mh n P eel Give we gere e e woe ds 3 3 About Quantitative Assays 20 n 3 4 What Is a Quantification Assay 2 0 0 0 eee 3 4 Instruments 2ce bel gbe3emEtbvevwewqpevehe cob degumd edu Rm enr ren RoE E NR 3 4 Terms Used in Quantification Analysis 0 e eee ee 3 4 How Real Time PCR Quantification Assays Work llle 3 6 Selecting a Quantification Assay Chemistry 0 00 e eee ee 3 6 Chemistries 3 d os we hy ad Pale Gene Wiha eee ee QVE a ELA P EX 3 6 Primers Used for cDNA Synthesis in Two Step RT PCR 2 0 000 3 6 Purchasing a Preformulated or Custom Designed Quantification Assay 3 7 Assays on Demand Gene Expression Product 200000 ee eee 3 7 Product Description en Se dats Ret ERE Ee uen eee Nae Sees 3 7 Purpose cic a ea leni oh MEG eto datae Be tea Shee aes gel 3 7 Product Properties lslllslllelleell res 3 8 Available Products 00 eee eee eee ee 3 8 About Target ASSays zz sois t SUELE EE SEED RU RUE Bead RH Lf xd 3 8 Assays by Design Service 00 e
103. ns or Master Mix may result in suboptimal performance If you follow the guidelines below the amplicons should be 50 to 150 basepairs By limiting the parameters for amplicon design such as amplicon size it is possible to run all reactions with a single reaction buffer such as TaqMan Universal PCR Master Mix and a single thermal cycling protocol Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more should be avoided The T of the primers should be 58 to 60 C Keep the G C content within 30 to 80 Make sure the last five nucleotides at the 3 end contain no more than two G C residues Place the forward and reverse primers as close as possible to the probe without overlapping it Selecting the Appropriate Reagent Configuration 4 8 Assays Containing TaqMan MGB Probes There are several TaqMan chemistry kits available for allelic discrimination assays The reagent configuration you use depends on your particular assay The reagent configurations listed in Table 4 3 are recommended for allelic discrimination assays These contain the TaqMan MGB probes Table 4 3 Reagent configurations for allelic discrimination assays Product Reagent Configuration Part Number TagMan TagMan Pre Developed Please see our web site for part Assay Reagents for Allelic numbers Keyword PDAR See Discrimination How to Obtain Support on page xi TaqMan Universal PCR 4324018
104. ocal requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis 1f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Be aware that high solvent flow rates 740 mL min may cause a static charge to build up on the surface of the tubing Elect
105. of a messenger RNA mRNA target for differences in the amount of total RNA added to each reaction e Exogenous control This is a characterized RNA or DNA spiked into each sample at a known concentration An exogenous active reference is usually an in vitro construct that can be used as an internal positive control IPC to distinguish true target negatives from PCR inhibition An exogenous reference can also be used to normalize for differences in efficiency of sample extraction or complementary DNA cDNA synthesis by reverse transcriptase Passive A dye that provides an internal reference to which the reporter dye reference signal is normalized Whether or not an active reference is used it is important to use a passive reference for example ROX dye in order to normalize for non PCR related fluctuations in fluorescence signal Normalized A unitless number that can be used to compare the relative amount of amount of target target in different samples CV Coefficient of variation The ratio of the standard deviation of a distribution to its arithmetic mean Calibrator In relative quantification the sample used as the basis for comparative results Endogenous Internal control gene present in each experimental sample By using an control endogenous control as an active reference you can normalize quantification of a messenger RNA mRNA target for differences in the amount of total RNA added to each reaction
106. oided When using Primer Express software the Tm should be 68 to 70 C When using Primer Express software the Tm should be 58 to 60 C No G on the 5 end Select the strand that gives the probe more C than G bases Make TaqMan MGB probes as short as possible without being shorter than 13 nucleotides The five nucleotides at the 3 end should have no more than two G and or C bases Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay Selecting the Appropriate Reagent Configuration There are several TaqMan Probe based and SYBR Green I dye chemistry kits available for Quantitative Assays The reagent configuration you use depends on your assay type The recommended reagent configurations are listed in the tables below A detailed discussion of the primary reagents follows on page 3 14 Recommended Reagent Configurations Note The part numbers listed below are for 200 reactions See Appendix C for a list of available kit sizes A number of different reagents are available for DNA and cDNA Quantification Assays Table 3 4 Reagents available for DNA and cDNA quantification assays Chemistry Reagent Configuration Part Number TaqMan probes TaqMan Universal PCR Master Mix 4304437 TaqMan Universal PCR Master Mix No AmpErase 4324018 UNG TaqMan PCR Core Reagents Kit N808 0228 SYBR Green dye SYBR Green PCR Master Mix 4309155 SYBR Green PCR Co
107. ol Baseline C Cycle Figure 3 1 Model of a single sample amplification plot showing terms commonly used in quantitative analysis How Real Time Real time PCR allows reactions to be characterized by the point in time during PCR Quantifica cycling when amplification of a PCR product achieves a fixed level of fluorescence tion Assays Work rather than the amount of PCR product accumulated after a fixed number of cycles An amplification plot graphically displays the fluorescence detected over the number of cycles that were performed As shown in Figure 3 1 in the initial cycles of PCR there is no significant change in fluorescence signal This predefined range of PCR cycles is called the baseline First the software generates a baseline subtracted amplification plot by calculating a mathematical trend using Rn values corresponding to the baseline cycles Then an algorithm searches for the point on the amplification plot at which the delta Rn value crosses the threshold The fractional cycle at which this occurs is defined as the C4 Selecting a Quantification Assay Chemistry Chemistries Quantification Assays can be used with the following chemistries e TaqMan Probe based fluorogenic 5 nuclease chemistry SYBR Green dye chemistry Both TaqMan Probe based and SYBR Green I dye chemistries can be used for either one step or two step RT PCR Primers Used for For two step RT PCR the following primers can be used for cDNA s
108. olymerase chain reaction PCR process Text Conventions This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow bracket 7 separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs User Attention Two user attention words appear in Applied Biosystems user documentation Each Words 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 chemistry kit use or safe use of a chemical Sequence Detection Systems Chemistry Guide ix Preface Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well microtiter plate Safety Alert Safety
109. on Getting Started Guide or the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION TRADEMARKS ABI PRISM Applied Biosystems GeneAmp Primer Express SYBR and VIC are registered trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries AB Design Applera Assays on Demand FAM MultiScribe ROX TAMRA and TET are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries Assays by Design is a service marks of Applera Corporation or its subsidiaries in the U S and or certain other countries AmpErase Amplitaq Gold and Taqman are registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4348358 Rev A 11 2003 Contents Preface How to Use This Guide bovis td whole eed hed Ree bei bee ew Seed ix How to Obtain More Information lees X How to Obtain Support ssis enie einn k nn a eee xi Safety and EMC Compliance Information Safety Conventions Used in This Document 20000 cee eee eee xiv General Instrument Safety llle XV Chemical Sael ysa aos aalg irai paa a Gh A an a cle Dee aaa XV Chemical Waste Safety eei eiaa oei p ae EE eee xvi Biological Hazard Safety 0 0 0 ie Dirar e i e E a E EA xviii Chapter 1 Introduction Select
110. on Systems and Assay Types Real Time Assays Endpoint Assays Instruments gt Gene Expression Big ee ARIRE 7900HT System Yes Yes No 7300 System Yes Yes Yes 7500 System Yes Yes Yes 7000 System Yes Yes Yes 7700 System Yes Yes Yes 5700 System Yes No No a The 5700 System cannot perform multiplex same tube reactions therefore the 5700 System cannot be used for quantification assays using multiplex PCR Allelic Discrimination Assays or Plus Minus Assays using an IPC Table 1 3 Consumable Types Supported Consumable Instruments HN 96 Well Micro Plate 384 Well Plate pene 7900HT System Yes Yes Yes 7300 System Yes No No 7500 System Yes No No 7000 System Yes No No 7700 System Yes No No 5700 System Yes No No a For more information on the MicroFluidic card see the 7900HT System User Guide About Real Time Real time PCR is the ability to monitor the progress of the PCR as it occurs Data is PCR Assays collected throughout the PCR process rather than at the end of the PCR process In real time PCR reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles Sequence Detection Systems Chemistry Guide 1 3 Chapter 1 Introduction 1 4 About One Step RT PCR RT PCR is used to quantify RNA RT PCR can be perf
111. ormed as a one step or two step procedure The one step RT PCR performs RT as well as PCR in a single buffer system Figure 1 1 The reaction proceeds without the addition of reagents between the RT and PCR steps This offers the convenience of a single tube preparation for RT and PCR amplification However the carryover prevention enzyme AmpErase UNG uracil N glycosylase cannot be used with one step RT PCR In one step RT PCR the presence of UNG would destroy the cDNA as it is being made For information about UNG see Using UNG to Minimize Reamplification Carryover Products on page 2 6 Primer extended on MRNA B V NN em MRNA RT er Tr cDNA everse Primer Synthesis of 1st cDNA strand M 5 CDNA Primer extended on cDNA 3 5 Forward Primer Synthesis of 2nd cDNA strand E D m PCR amplification of CDNA Forward Primer Reverse Primer GR0746 Figure 1 1 Schematic representation of one step RT PCR About Two Step RT PCR Two step RT PCR is performed in two separate reactions Figure 1 2 This is useful when detecting multiple transcripts from a single cDNA reaction or when storing a portion of the cDNA for later use When performing PCR if dUTP is not used as a base in the RT step AmpErase UNG enzyme can be used to prevent carryover contamination Sequence Detection Systems Chemistry Guide About Endpoint Assays About Multiplex PCR Selecting an Assay Type Prim
112. out being shorter than 13 nucleotides Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be avoided Position the polymorphic site in the central third of the probe Note The polymorphic site can be shifted toward the 3 end to meet the above guidelines however the site must be located more than two nucleotides upstream from the 3 terminus Figure 4 2 illustrates the placement of a polymorphism in an example probe N Nucleotide Polymorphism If necessary place the polymorphism here 5 3 N NN N N N N N N NNN NN N N N NN NN First try to position the polymorphic Do not place the site in the central third of the probe polymorphism here Figure 4 2 Polymorphism placement in an example probe Sequence Detection Systems Chemistry Guide 4 7 Chapter 4 Allelic Discrimination Assays Primer Design Using Primer Express Software Primer Design Guidelines The Primer Express software uses a set of default parameters to automatically select primer and probe sets The primer design guidelines for allelic discrimination assays are discussed here For more information refer to Primer Express Software Version 2 0 Users Manual PN 4329500A After selecting probes for the assay see page 4 6 choose primers based on the guidelines below Note Primer Express software is designed for universal assay conditions Changing assay conditio
113. pecific PCR products Real time systems for PCR were improved by the introduction of fluorogenic labeled probes that use the 5 nuclease activity of AmpliTaq Gold DNA polymerase The availability of these fluorogenic probes enabled the development of a real time method for detecting only specific amplification products The TaqMan Probe based chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR Here s how it works l An oligonucleotide probe is constructed with a fluorescent reporter dye bound to the 5 end and a quencher on the 3 end While the probe is intact the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer FRET F rster resonance Forster V T 1948 through space If the target sequence is present the probe anneals between primer sites and is cleaved by the 5 nuclease activity of AmpliTaq Gold polymerase during extension This cleavage of the probe Separates the reporter dye from the quencher increasing the reporter dye signal Removes the probe from the target strand allowing primer extension to continue to the end of the template strand Thus inclusion of the probe does not inhibit the overall PCR process Additional reporter dye molecules are cleaved from their respective probes with each cycle resulting in an increase in fluorescence intensity propo
114. phism screening letter Nat Genet 9 341 342 Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Sequence Detection Systems Chemistry Guide B 1 Appendix B References B 2 Sequence Detection Systems Chemistry Guide Part Numbers This chapter covers Sequence Detection Instruments lisse ees C 2 Sequence Detection Kats ues ete Ce eru CER de DEO SEE e NE C 3 Sequence Detection PCR Reagent Kits 0 0 0 c cee eese C 4 Sequence Detection RT PCR Kits 0 cette C 6 Sequence Detection Reaction Kits with Controls 00 0 e ce eee C 7 Sequence Detection Control Reagents 0 0 cece eee eens C 8 Sequence Detection Reagent Components cece cece eens C 9 Sequence Detection Calibration Kits 00 0 usann eens C 10 Sequence Detection Disposables 0 0 0c cece eee eens C 11 Custom Oligonucleotide Synthesis 0 0 20 0 0 cece cece eens C 13 Sequence Detection Systems Chemistry Guide C 1 Appendix C Part Numbers Sequence Detection Instruments Part Number Instrument 4329002 ABI PRISM 7900HT Real Time PCR System with 384 Well Block Module and Automation Accessory 4329004 ABI PRISM 7900HT Real Time PCR System with 96 Well Block Module and Automation Accessory 4329001 ABI PRISM 7900HT Real Time PCR System with
115. pplied Biosystems has four instruments in its Sequence Detection System SDS product line Applied Biosystems 7900HT Real Time PCR System 7900HT System Applied Biosystems 7300 Real Time PCR System 7300 System Applied Biosystems 7500 Real Time PCR System 7500 System ABI PRISM 7000 Sequence Detection System 7000 System ABI PRISM 7700 Sequence Detection System 7700 System GeneAmp 5700 Sequence Detection System 5700 System Note For more detailed information on these instruments refer to the instrument user guides See How to Obtain More Information on page x for a list of the user guide titles and part numbers Assay Types The three assay types quantification allelic discrimination and plus minus can be Supported divided into two categories as shown in Table 1 1 Table 1 1 Assay types Real Time PCR For Information See Endpoint Assay For Information See Quantification including About Quantitative Allelic Discrimination About Allelic One step reverse Assays on page 3 4 Discrimination Assays on transcription page 4 2 rdc EEE ECB Dor Plus Minus About Plus Minus Assays RNA quantification Using an IPC on page 5 2 Two step RT PCR for RNA quantification DNA quantification 1 2 Sequence Detection Systems Chemistry Guide Selecting an Assay Type The sequence detection systems can be used to perform the assay types in Table 1 2 Table 1 2 Sequence Detecti
116. ption to determine the amount of RNA added to a cDNA reaction You then run a PCR using cDNA derived from the same amount of input RNA You can use this approach to select an appropriate endogenous control and to determine if an endogenous control is affected by the treatment In this case the target gene and the endogenous reference are the same item Livak and Schmittgen 2001 Standards As the sample quantity is divided by the calibrator quantity the unit from the standard curve drops out Thus all that is required of the standards is that their relative dilutions be known For relative quantification this means any stock RNA or DNA containing the appropriate target can be used to prepare standards Sequence Detection Systems Chemistry Guide 3 29 Chapter 3 Gene Expression and Other Quantitative Assays How to Perform the Relative Standard Curve Method Example of the Relative Standard Curve Method 3 30 To perform the relative standard curve method for quantification Perform a run on your SDS instrument this includes Setting up a reaction plate Analyzing the data Creating a standard curve Determine the relative values See Example of the Relative Standard Curve Method below for an illustration of these steps This example illustrates the use of standard curves for relative quantification based on The target is human c myc mRNA and the endogenous control is human GAPDH mRNA The target and
117. rameters Recommended Thermal Cycling Parameters Sequence Detection Systems Chemistry Guide All Quantitative Assays designed using Applied Biosystems Assay Design Guidelines can be run using the universal thermal cycling parameters This eliminates any optimization of the thermal cycling parameters and means that multiple assays can be run on the same plate without sacrificing performance This benefit is critical when combining two assays into a multiplex 5 nuclease assay system DNA cDNA Quantification The thermal cycling parameters listed in Table 3 7 are recommended for DNA and cDNA Quantification Assays The parameters apply to both TaqMan Probe based and SYBR Green I dye chemistries Table 3 7 Thermal cycling parameters for DNA and cDNA quantification assays Times and Temperatures Initial Steps PCR Each of 40 Cycles AmpliTaq Gold ae DNA Polymerase Melt Anneal Extend Activation HOLD HOLD CYCLE 2 min 50 C 10 min 95 C 15 sec 95 C 1 min 60 C RNA Quantification Using One Step RT PCR The thermal cycling parameters listed in Table 3 8 are recommended for RNA Quantification Assays using one step RT PCR The parameters apply to both TaqMan Probe based and SYBR Green I dye chemistries Table 3 8 Thermal cycling parameters for RNA quantification assays using one step RT PCR Times and Temperatures Initial Steps PCR Each of 40 Cycles AmpliTaq Gold iare DNA
118. re Reagents 4304886 RNA Quantification Using One Step RT PCR A number of reagents are available Table 3 5 for RNA Quantification Assays using one step RT PCR Table 3 5 Reagents available for RNA quantification assays using one step RT PCR Chemistry Reagent Configuration Part Number TaqMan probes TaqMan One Step RT PCR Master Mix Reagents 4309169 Kit TaqMan Gold RT PCR Kit without controls N808 0232 TaqMan EZ RT PCR Core Reagents N808 0236 Note Use this configuration when a high temperature RT step is required SYBR Green dye SYBR Green RT PCR Reagents 4310179 Sequence Detection Systems Chemistry Guide 3 13 Chapter 3 Gene Expression and Other Quantitative Assays About Universal 3 14 Master Mix Reagents About the Reagent Components RNA Quantification Using Two Step RT PCR A number of reagents are available Table 3 6 for RNA Quantification Assays using two step RT PCR Table 3 6 Reagents available for RNA quantification assays using two step RT PCR Chemistry Step Reagent Configuration Part Number TaqMan PCR step only TaqMan Universal PCR Master Mix 4304437 probes RT step only TaqMan Reverse Transcription N808 0234 Reagents High Capacity cDNA Archive Kit 4322171 Both RT and TaqMan Gold RT PCR kit N808 0232 PCR steps SYBR Green PCR step only SYBR Green Master Mix 4309155 dye Both RT and SYBR Green RT PCR Reagents 4310179 PCR s
119. rently available check the web site at a later time or use our Assays by Design M service see below Assays on Demand Gene Expression Products are designed for the detection and quantification of specific nucleic acid sequences These products provide researchers with optimized ready to use 5 nuclease assays for human and mouse transcripts Gene expression quantification is performed in a two step RT PCR in which the PCR step is coupled with a 5 fluorogenic nuclease assay Sequence Detection Systems Chemistry Guide 3 7 Chapter 3 Gene Expression and Other Quantitative Assays Product The following are properties of Assays on Demand Gene Expression Products Properties All Assays on Demand Gene Expression Products are designed and optimized to work with the TaqMan Universal PCR Master Mix with or without AmpErase UNG A variety of targets and endogenous controls can be assayed in singleplex reactions in the same reaction plate with universal thermal cycling parameters The Assays on Demand Gene Expression Products are designed to amplify target cDNA without amplifying genomic DNA Assays denoted m in the assay name for example Hs00265057 m1 are designed so that the probe spans an exon exon junction for multi exon genes Assays denoted s in the assay name for example Hs00257856 s1 are designed within an exon and by definition will detect genomic DNA Available Assays on Demand Gene Expression Products PN
120. representation of how accurately the multicomponented data fits the raw data The higher the MSE value the greater the deviation between the multicomponented data and the raw data When using the 7700 and 7900HT systems ensure that the plate is set up correctly and the detectors are labeled 6 2 Sequence Detection Systems Chemistry Guide Table 6 1 Troubleshooting Quantification Assays Troubleshooting analyzed run data quantification assays continued Analysis View Description Look For Amplification Plot Displays data from real time runs after signal normalization and multicomponent analysis It contains the tools for setting the baseline and threshold cycle C4 values for the run Correct baseline and threshold settings Are the baseline and threshold values set correctly Identify the components of the amplification curve Determine the cycle for which amplification first begins to be distinquishable from the noise Set the baseline so that the endpoint is 1to 2 cycles before this point Identify the components of the amplification curve and set the threshold so that it is e Above the background Below the plateaued and linear regions Within the geometric phase of the amplification curve Irregular amplification Do all samples appear to have amplified normally with a smooth amplification plot free of sharp spikes or dips The three phases of the amplification curve should be clearly vis
121. rical sparks may result Sequence Detection Systems Chemistry Guide xvii Safety and EMC Compliance Information Biological Hazard Safety xviii General Biohazard ANIE BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Additional information about biohazard guidelines is available at http www cdc gov Sequence Detection Systems Chemistry Guide Introduction This chapter covers Selecting an Assay Type 0 cece tte e eens 1 2 Selecting the Sequence Detection Chemistry 0 2 0 c eee ee eee 1 6 Selecting an Assay Source 0 ete t teens 1 7 Performing the Assay sce soe Ve PU a ea d e oe aes 1 9 Selecting a Data Analysis Approach and Determining Results 1 9 Sequence Detection Systems Chemistry Guide 1 1 Chapter 1 Introduction Selecting an Assay Type SDS Instruments A
122. rmation contact your Applied Biosystems representative 3 8 Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay Designing Your Own Quantification Assay Important Design This section discusses the Applied Biosystems Assay Design Guidelines developed Steps specifically for Quantification Assays These guidelines contain the following important steps Designing primers and probes using Primer Express software Selecting the appropriate reagent configuration Using universal thermal cycling parameters Using default primer and probe concentrations or optimizing if necessary IMPORTANT These steps provide a rapid and reliable system for assay design and optimization only when used in their entirety The system must be adopted as a whole in order to achieve the highest level of success due to the interdependence of many of the individual components Conclusions The Applied Biosystems Assay Design Guidelines enable Quantitative Assays to be designed and optimized rapidly and efficiently Since thousands of assays have been developed this way the following conclusions can be made For the vast majority of 5 nuclease quantification assays designed and run following these guidelines using a concentration of 900 nM primers and 250 nM probe will provide for a highly reproducible and sensitive assay when using DNA or cDNA as a template Due to the nonspecific nature of its detection SYBR Green I
123. rom 50 to 250 nM Sequence Detection Systems Chemistry Guide 3 21 Chapter 3 Gene Expression and Other Quantitative Assays Using Multiplex PCR Multiplex PCR is the use of more than one primer probe set in the same tube Multiplex PCR is most commonly used in 5 nuclease assays for the relative quantification of gene expression Typically one probe labeled with FAM dye is used to detect the target species another probe labeled with VIC dye is used to detect an endogenous control internal control gene Running both assays in a single tube reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes Multiplex in In order to multiplex using the comparative C method you must ensure that the Contrast to endogenous control that you have selected is more abundant lower C4 than all of the Singleplex targets that you are trying to quantify under all conditions and then you must run the endogenous control assay as a primer limited assay For multiplexing the endogenous control assay for the more abundant template in each reaction must be primer limited to avoid competitive PCR that may alter the Cy of the less abundant template Primer limited endogenous control reactions are used in multiplex assays to normalize for the amount of input nucleic acid into different wells as well as to normalize for effects on the target assay in the same well However a primer limited assa
124. rtional to the amount of amplicon produced The higher the starting copy number of the nucleic acid target the sooner a significant increase in fluorescence is observed Figure 2 2 illustrates this process Strand Displacement Cleavage Polymerization Completed gt Q Ro INO XK e 3 5 3 5 5 3 5 Step 1 continued When both dyes are attached to the probe reporter dye emission is quenched 5 x 5 Step 2 During each extension cycle the AmpliTaq Gold DNA polymerase cleaves the reporter dye from the probe 3 5 3 5 Step 3 Once separated from the quencher the reporter dye emits its characteristic fluorescence Figure 2 2 Representation of how the 5 nuclease chemistry uses a fluorogenic probe to enable detection of a specific PCR product Sequence Detection Systems Chemistry Guide 2 3 Chapter 2 Chemistry Overview Two Types of Applied Biosystems offers two types of TaqMan probes TaqMan Probes TaqMan probes with TAMRA dye as quencher e TaqMan MGB minor groove binder probes with non fluorescent quencher NFQ Table 2 1 describes each probe Table 2 1 Products Custom Product 5 Label 3 Label Other Features TaqMan probe FAM TET or VIC TAMRA TaqMan MGB FAM TET or VIC Nonfluorescent quencher minor groove binder Assays on Demand TaqMan MGB quencher Gene
125. ry Crp the difference in threshold cycles for target and reference Sequence Detection Systems Chemistry Guide Comparative CT Method for Relative Quantification Rearranging gives the following expression AC Xy Kx 1 E The final step is to divide the Xy for any sample q by the Xy for the calibrator cb AC fug _ Kx E AC AN KX L4H 7 E qup where AAC ACtg ACrop For amplicons designed and optimized according to Applied Biosystems Assay Design Guidelines amplicon size lt 150 bp the efficiency is close to 1 Therefore the amount of target normalized to an endogenous control and relative to a calibrator is given by 2 AACT Sequence Detection Systems Chemistry Guide A 3 Appendix A Formulas A 4 Sequence Detection Systems Chemistry Guide References Afonina I Zivarts M Kutyavin I et al 1997 Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder Nucleic Acids Res 25 2657 2660 Collins M L Zayati C Detmar J J Daly B Kolberg J A Cha T A Irvine B D Tucker J and Urdea M S 1995 Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays Anal Biochem Mar 20 226 120 129 Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann Physics Leipzig 2 55 75 Higuchi R Dollinger G Walsh P S and Griffith
126. s When faced with irregular data you can use the SDS software to diagnose some chemistry and instrument related problems Table 6 3 contains a summary of checks to verify the integrity of your run data and to help you begin troubleshooting Analysis View Description Look For Recommended Action Raw Data Displays the composite fluorescence signal not normalized for the selected wells during each cycle of the PCR Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate or sample block could be contaminated Characteristic signal shape Do the samples peak at the expected wavelengths Signal plateaus Do any of the signals plateau Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal Clean the contaminated block and generate new samples Repeat the experiment Sequence Detection Systems Chemistry Guide 6 7 Chapter 6 Troubleshooting 6 8 Sequence Detection Systems Chemistry Guide Formulas Comparative C4 Method for Relative Quantification Formula The amount of target normalized to an endogenous control and relative to a calibrator is given by 2 AACT Derivation of the The equation that describes the exponential amplification of PCR is Formula X X x 14 E where X number of target molecules at cycle n
127. s required for SYBR Green I dye detection it is still a good idea to use Primer Express software to select a primer and probe set when designing a SYBR Green I dye assay Although no probe will be used the primers will meet all the required criteria and if in the future there is the need to convert the assay to 5 nuclease assay chemistry to obtain higher specificity the probe can immediately be found in the original Primer Express software document Selecting a good amplicon site ensures amplification of the target mRNA cDNA without co amplifying the genomic sequence pseudogenes and other related genes SYBR Green I dye chemistry can be useful for screening amplicon sites for gene expression Guidelines The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA The primer pair should be specific to the target gene to avoid amplification of pseudogenes or other related genes When designing primers use Primer Express guidelines fno good sequence is found it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites If the gene you are studying does not have introns then it is not possible to design an amplicon that will amplify the mRNA sequence without amplifying the gene sequence In this case it is necessary to run RT minus controls Selection of Small Amplicons An important default parameter in Primer Express software is the sel
128. se steps provide a rapid and reliable system for assay design and optimization only when used in their entirety The system must be adopted as a whole in order to achieve the highest level of success due to the interdependence of many of the individual components Sequence Detection Systems Chemistry Guide Performing the Assay To illustrate this point consider the following example The ability to use universal thermal cycling parameters is based on the assumption that the selected primers have a melting temperature T a of 58 to 60 C as calculated by Primer Express software If the primers do not have the correct Ts or even if the T s have been calculated with a primer design software package other than Primer Express optimal performance and even functionality of the assay cannot be assured Visit the Applied Biosystems web site to access a variety of tutorials on how to use the Primer Express Software for designing real time quantitative assays See How to Obtain Support on page xi Performing the Assay For information about performing the assay on your system see the documentation provided with your system For information about laboratory practices see Minimizing DNA Contaminants on page 2 6 Selecting a Data Analysis Approach and Determining Results For information see Section 3 2 Selecting a Data Analysis Approach and Determining Results Sequence Detection Systems Chemistry Guide 1 9 Chapter 1 Introductio
129. stry Guide Chapter 6 Troubleshooting Troubleshooting Quantification Assays Using SDS When faced with irregular data you can use the SDS software to diagnose some Software to chemistry and instrument related problems Figure 6 1 on page 6 6 contains a Diagnose summary of checks to verify the integrity of your run data and to help you begin Irregular Data troubleshooting potential problems Table 6 1 Troubleshooting analyzed run data quantification assays Analysis View Description Look For Raw Data Plot Displays the composite raw fluorescence signal not normalized for the selected wells during each cycle of PCR Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate or sample block could be contaminated Characteristic signal shape Do the samples peak at the expected wavelengths For example samples containing only FAM dye labeled TaqMan probes should not produce raw fluorescence in the wavelength of a VIC dye component A signal present in wells that do not contain the dye could indicate that the sample master mix or well contains contaminants Characteristic signal growth As you drag the bar through the PCR cycles do you observe growth as expected Absent growth curves may indicate a pipetting error well lacks template or no amplification Signal Plateaus Do any of the signals plateau Signal platea
130. stry Guide 3 25 Chapter 3 Gene Expression and Other Quantitative Assays Data Analysis General Process Resources for Data Analysis Data analysis varies depending on the product assay and instrument Refer to the appropriate instrument user guide for instructions on how to analyze your data The general process for analyzing the data from gene expression assays involves the following procedures 1 Viewing the amplification plots for the entire plate 2 Setting the baseline and threshold values 3 Using the methods detailed in this section to determine results For more information about analyzing your data see Livak and Schmittgen 2001 Also refer to the following documents The appropriate instrument user guide User Bulletin 2 Relative Quantification of Gene Expression PN 4303859 Data Analysis and Relative Quantification chapters in the TaqMan Cytokine Gene Expression Plate I Protocol PN 4306744 This protocol provides examples using multiplex reactions RQ Manager Software User Guide PN 4339753 ABI PRISM 7900HT Real Time PCR System User Guide PN 4317596 SDS Enterprise Database Administration Guide PN 4346513 Note Some documents are available through the Internet see How to Obtain Support on page xi Relative or Absolute Quantification What Is Relative Quantification 3 26 You can design your assay as relative or absolute quantification How you calculate the results of your
131. t words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical iz Ye Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices rom ndicates a potentially hazardous situation that 1f not avoided could result in death or serious injury Deli 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 Applied Biosystems instruments Examples The following examples show the use of safety alert words IMPORTANT You must create a separate a Sample Entry Spreadsheet for each 96 well plate ANITIN The lamp is extremely hot Do not touch the lamp until it has cooled to room temperature Nee CHEMICAL HAZARD Formamide Exposure causes eye
132. taining Accurate Quantification For the most accurate quantification using two probes in one tube use the reporter dyes that have the largest difference in emission maximum FAM and VIC dye Reactions designed to amplify two different sequences in the same tube share common reagents If the two sequences have different initial copy numbers it is possible for the more abundant species to use up these common reagents impairing amplification of the rarer species For accurate quantification it is important that the two reactions do not compete Competition can be avoided by limiting the concentration of primers used in the amplification reactions For more information on primer limitations see page 3 23 Sequence Detection Systems Chemistry Guide Multiplex PCR Same Tube Method for Relative Quantification How to Perform To perform the multiplex PCR with the relative standard curve method for Multiplex PCR quantification with the Relative Standard Curve Method Perform arun on your SDS instrument This includes Setting up a reaction plate Analyzing the data Creating a standard curve Determine the relative values See Example of Multiplex PCR with the Relative Standard Curve Method below for an illustration of these steps Example of This example illustrates the use of multiplex PCR with the relative standard curve Multiplex PCR method for quantification In this example with the Relative e The target is hum
133. teps TaqMan Universal PCR Master Mix The TaqMan Universal PCR Master Mix is a reagent specifically designed to provide optimal performance for 5 nuclease assays that use cDNA or DNA as a template This product contains components that ensure excellent assay performance even when demanding G C rich target sequences are encountered The use of one reagent for all assays simplifies the process of assay implementation SYBR Green PCR Master Mix The SYBR Green PCR Master Mix is a convenient premix for real time PCR using the SYBR Green I dye Direct detection of the PCR product is monitored by measuring the increase in fluorescence that is caused by the SYBR Green I dye binding to double stranded DNA AmpliTaq Gold DNA Polymerase The use of the hot start enzyme AmpliTaq Gold DNA Polymerase is an integral part of Applied Biosystems Assay Design Guidelines for both TaqMan Probe based and SYBR Green I dye chemistries The use of AmpliTaq Gold DNA Polymerase ensures a robust reaction and can dramatically reduce the amount of nonspecific product formation A further benefit is the simplification of assay setup which can be performed at room temperature MultiScribe Reverse Transcriptase MultiScribe Reverse Transcriptase is a recombinant Moloney Murine Leukemia Virus MuLV Reverse Transcriptase Sequence Detection Systems Chemistry Guide Designing Your Own Quantification Assay Using the Universal Thermal Cycling Pa
134. ter 6 Troubleshooting Troubleshooting Allelic Discrimination Assays Troubleshooting DNA Concentration Effects Run Data Observation Scattering of data points Possible Causes Very low DNA concentrations or variable concentrations Figure 6 1 shows the benefits of using relatively high DNA concentrations Using relatively high DNA concentrations yields large ARn As can be seen in Figure 6 2 on page 6 7 at 1 ng 5 ng and 20 ng within a DNA amount the cluster is tight and easily scorable The data presented in Figure 6 2 was generated by running the samples at low to high concentrations If all samples have relatively high at least 1 ng DNA concentration the clusters are scorable When samples have very low DNA concentrations such as 0 01 and 0 1 clusters become diffuse making scoring difficult 2 000 1 800 1 600 1 400 1 200 g 1 000 4 8 000 E1 6 000 E1 4 000 E1 2 000 E1 Figure 6 1 Effects of DNA concentration on ARn 6 6 Sequence Detection Systems Chemistry Guide Troubleshooting Allelic Discrimination Assays Normalized FAM e 20ng m NTC A 5ng x Ing x 0 1ng e 0 01ng Normalized VIC Figure 6 2 Effects of different DNA concentrations Recommended Action Verify that samples all have relatively high concentrations Troubleshooting Analyzed Run Data potential problems Table 6 3 Troubleshooting analyzed run data allelic discrimination assay
135. ultiple reporter dyes 3 40 multiplex accuracy reporter dyes 3 40 multiplex assays rRNA primers 3 23 Sequence Detection Systems Chemistry Guide multiplex PCR and relative quantification 3 40 comparative CT example 3 43 comparative CT results 3 44 described 1 5 multiple reporter dyes 3 40 primer limiting 3 23 relative standard curve 3 41 relative standard curve example 3 41 singleplex comparison 3 22 using 3 22 MultiScribe reverse transcriptase defined 3 14 N nonspecific product contamination with SYBR dye 2 6 Notes description ix O one step RT PCR and RNA quantification 3 13 optimization allelic discrimination assays 4 6 probe concentration 3 19 P PCR general practices 2 7 plus minus assay and TaqMan chemistry 5 3 defined 5 2 incorporating an IPC 5 3 instruments 5 2 internal positive control IPC 5 2 process described 5 3 reagent configurations 5 5 terms described 5 3 primer concentrations defaults 3 17 primer design guidelines 4 8 Primer Express software and assay design 3 9 and primer design 4 8 and small amplicons 3 10 primer limiting multiplex assays 3 23 primer matrix and defining limits 3 23 example of limiting 3 24 primer optimization matrix how used 3 17 primers and hairpin loops 3 7 limitations 3 40 probe concentration optimizing 3 19 recommendations 3 20 Sequence Detection Systems Chemistry Guide probe design allelic discrimination assay 4 7 probes custom products listed 2 4
136. us or saturation can be an indication that a well contains too much template or fluorescent signal The 7700 instrument saturates at 32 500 fluorescent units The 7900HT instrument saturates at 66 000 fluorescent units Multicomponent Plot Displays a plot of normalized multicomponent data from a single well of a real time run The plot displays the component dye signals that contribute to the composite signal for the well Correct dyes displayed Does the plot display all dyes as expected The presence of an unexpected dye may be the result of an error in detector setup such as assigning the wrong reporter or quencher dye ROX dye fluorescence level Does the ROX dye signal fluoresce below the reporter dyes If not the lack of reporter fluorescence may be caused by an absence of probe in the well a pipetting error A drop in ROX dye fluorescence level can be caused by non homogenous mix evaporation primer ROX dye interaction cover not seated properly Note A slight dip in ROX dye may occur after Cycle 35 This is normal Reporter dye fluorescence level Does the reporter dye signal fluoresce above background The background signal is a measure of ambient fluorescence If a dye fails to fluoresce above the background it is a strong indication that the well is missing probes labeled with the dye well does not contain probe PCR master mix or both MSE Level The MSE mean squared error is a mathematical
137. ve Efficiency of Target and Reference 0 0c eee eee eee 3 37 How to Perform the Comparative CT Method 0000 e cease 3 37 Example of the Comparative CT Method 2000 0c eee eee eee 3 37 Multiplex PCR Same Tube Method for Relative Quantification 3 40 Advantages of Multiplex PCR s ecceri eiia ne eE ete 3 40 About Multiple Reporter Dyes 000 cee eee 3 40 About Multicomponenting 0 cece ee Rh 3 40 About Primer Limitation llle 3 40 How to Perform Multiplex PCR with the Relative Standard Curve Method 3 41 Example of Multiplex PCR with the Relative Standard Curve Method 3 41 How to Perform Multiplex PCR with the Comparative CT Method 3 43 Example of Multiplex PCR with the Comparative CT Method 3 43 Standard Curve Method for Absolute Quantification 2000006 3 46 Requirements 5 vd sre ght snaked eating eet MESURE AREE FREE 3 46 Standards uice exa e ORDRE EPMOQEE ve cout Feds Pu E EVI AEN 3 46 How to Perform the Absolute Standard Curve Method s 3 46 Sequence Detection Systems Chemistry Guide V Chapter 4 Chapter 5 vi Allelic Discrimination Assays About Allelic Discrimination Assays lise eee 4 2 What Is an Allelic Discrimination Assay llle 4 2 Instruments i eae hee ed ele eae ee eles Malgeg Ted Sema REPE wal 4 2 Chemisty ib re Peek eed 6 oy seen SER
138. y A269 and converted to the number of copies using the molecular weight of the DNA or RNA Except for preparation of the standards see above the absolute standard curve methods and relative standard curve quantification methods are identical To perform the absolute standard curve method for quantification see How to Perform the Relative Standard Curve Method on page 3 30 Example of the Relative Standard Curve Method on page 3 30 Sequence Detection Systems Chemistry Guide Allelic Discrimination Assays This chapter covers About Allelic Discrimination Assays 0 0000s ccc ct enn 4 2 Purchasing an Applied Biosystems Preformulated or Custom Designed Allelic Discrimination Assay serete ior ta Eo KEETA ehe 4 4 Designing Your Own Allelic Assay lesse 4 6 Sequence Detection Systems Chemistry Guide 4 1 Chapter 4 Allelic Discrimination Assays About Allelic Discrimination Assays What Is an Allelic Discrimination Assay Instruments Chemistry Terms Used in Allelic Discrimination Analysis An Allelic Discrimination Assay is an endpoint assay used to determine the genotype of samples With this assay type it is possible to differentiate a single nucleotide polymorphism SNP The aim of an Allelic Discrimination Assay is to classify unknown samples as Homozygotes samples having only allele 1 Homozygotes samples having only allele 2 Heterozygotes samples having both allele 1 and alle
139. y Guide 2 5 Chapter 2 Chemistry Overview Minimizing DNA Contaminants Using UNG to Minimize Reamplification Carryover Products The DNA amplification capability of the PCR process makes special laboratory practices necessary when performing assays using fluorogenic 5 nuclease TaqMan Probe based chemistry or SYBR Green I dye chemistry Potential contamination can be introduced by samples with high DNA concentrations either from the DNA template controls or from PCR carryover In addition due to the nonspecific nature of the SYBR Green I dye any double stranded DNA will be detected When using the SYBR Green I dye chemistry check for nonspecific product formation by using dissociation curve or gel analysis Care must be taken to avoid contamination with target DNA Gene expression assays that span exon exon junctions minimize the effect of gDNA genomic DNA contaminants Note The ABI PrisM 7700 Dissociation Curve Set up Quick Start Guide is available through the Applied Biosystems web site see How to Obtain Support on page xi AmpErase uracil N glycosylase UNG is a 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene This gene has been inserted into an E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The en
140. y may be more susceptible to fluctuations in reaction conditions than the primer non limited target assay that it is normalizing Doing this in a multiplex format becomes increasingly more complex as the number of targets you wish to quantify increases It becomes increasingly unlikely that you will be able to identify a suitable endogenous control that will be more abundant than all of the targets you wish to query and whose expression does not change as a result of the experimental conditions or across different samples As a result for an increasing number of targets it is likely to be more efficient and effective to run the targets and controls in the singleplex format utilizing the precision of the sequence detection system in conjunction with delivery of equivalent amounts of input material to different reaction wells For these reasons when you analyze multiple numbers of targets it 1s likely to be more effective to run your assays in the singleplex format To multiplex you would have to first run all of your target assays and endogenous control assays in both the multiplex and singleplex format and compare Cy values from both formats to determine if there are any effects of the multiplexing on your Cy values which could be a larger undertaking than the study itself Also by using the singleplex method any target can potentially be used as an endogenous control at the analysis step of the process Any target whose expression level does
141. yc GAPDH 100 0 20 28 0 11 15 23 0 02 5 05 0 11 10 0 23 88 0 14 18 62 0 04 5 26 0 15 1 0 27 33 0 09 22 18 0 02 5 15 0 09 0 1 30 93 0 23 25 53 0 07 5 40 0 24 0 01 34 39 0 37 29 13 0 14 5 26 0 39 0 001 37 03 0 12 32 57 0 13 4 46 0 17 Relative For the AAC calculation to be valid the efficiency of the target amplification and Efficiency of the efficiency of the reference amplification must be approximately equal A Target and sensitive method for assessing if two amplicons have the same efficiency is to look at Reference how AC varies with template dilution The standard curves for c myc and GAPDH used in the previous section provide the necessary data Table 3 15 shows the average C4 value for c myc and GAPDH at different input amounts How to Perform To perform the comparative C method for relative quantification the Comparative C Method Perform a run on your SDS instrument This includes Setting up a reaction plate Analyzing the data Determine the AC value Target Endogenous control Perform the AAC calculation to determine fold difference in gene expression AC Target AC Calibrator See the example on below for an illustration of these steps Example of the This example illustrates the use of the comparative C4 method for relative Comparative C4 quantification In this example Method The target is human c myc mRNA and the endogenous control is human
142. ynthesis cDNA Synthesis Oligo d T n bores Random hexamers Sequence specific reverse primers 3 6 Sequence Detection Systems Chemistry Guide Purchasing a Preformulated or Custom Designed Quantification Assay The choice of primers for reverse transcription is best made after experimentally evaluating all three priming systems For short RNA sequences containing no hairpin loops any of the three priming systems works equally well For longer RNA transcripts or sequences containing hairpin loops consider the guidelines in Table 3 2 Table 3 2 Priming systems for RT PCR Primers Selection Guidelines Oligo d T 4s e Use to reverse transcribe only eukaryotic mRNAs and retroviruses with poly A tails e Avoid long mRNA transcripts or amplicons greater than 2 kilobases upstream Random hexamers Try first for use with long reverse transcripts or reverse transcripts containing hairpin loops e Use to transcribe all RNA rRNA mRNA and tRNA Sequence specific e Use to reverse transcribe RNA containing complementary reverse primers sequences only Purchasing a Preformulated or Custom Designed Quantification Assay Assays on Demand Gene Expression Product Product Description Purpose Assays on Demand products for gene expression are biologically informative preformulated gene expression assays that provide rapid reliable results on human and mouse transcripts If the assay you need is not cur
143. zyme causes the release of uracil thereby creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 TaqMan Assays For 5 nuclease assays which use the TaqMan Universal PCR Master Mix AmpErase UNG treatment can prevent the re amplification of carryover PCR products When dUTP replaces dTTP in PCR amplification AmpErase UNG treatment can remove up to 200 000 copies of amplicon per 50 uL reaction SYBR Green Dye Assays AmpErase UNG treatment can also be useful in preventing the re amplification of carryover PCR products in SYBR Green I dye assays Although the SYBR Green PCR Master Mix does not contain AmpErase UNG dTTP has been replaced with dUTP thus making the SYBR Green PCR Master Mix compatible with the use of AmpErase UNG If contamination from PCR carryover is suspected use AmpErase UNG to troubleshoot the problem Note AmpErase UNG can be purchased individually PN N808 0096 or as part of the SYBR Green PCR Core Reagents kit PN 4304886 Sequence Detection Systems Chemistry Guide Minimizing DNA Contaminants General PCR Use the following precautions to minimize sample contamination and PCR product Practices carryover Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are

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