Sample & Assay Technologies RT2 qPCR Primer
Contents
1. Expression is seen when it is not expected a Genomic or exogenous DNA contamination 20 Perform and interpret appropriate negative control reactions NRT and NTC controls RT qPCR Primer Assay Handbook 03 2011 Comments and suggestions b Knockout experiment Expression may be detected if the RT qPCR Primer Assay is being used to validate a knockout mouse model where only a portion of the endogenous gene is replaced and the RT qPCR Primer Assay is not located in the replaced sequence of the resulting mRNA transcript Do not use RT qPCR Primer Assays for this purpose No template control NTC shows a C value 35 cycles a DNA contamination of See Preparing a workspace free of DNA reagents tips and contamination page 9 tubes b Presence of primer Verify the presence of primer dimers by agarose dimers gel electrophoresis primer dimmers are 50 bp in size Use the appropriate RT SYBR Green Mastermix to prevent the appearance of primer dimers RT qPCR Primer Assay Handbook 03 2011 21 Appendix A Data Analysis AAC method The AAC method is recommended for data analysis Perform the AAC method as described below In separate reactions determine the Cy value for the housekeeping gene s HKG and for the genes of interest GOI in each sample Only use C values less than 35 Only compare C values determined using the same amount of template For example Control C GOl 24 25 C HKG 12. If using plates instead of tubes centrifuge the plate for 1 min at 1000 g to remove bubbles 4 Program the real time cycler according to Table 4 5 or 6 depending on the real time cycler used Run the program Note For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarrayprotocolfiles php FE A 16 RT qPCR Primer Assay Handbook 03 2011 Table 4 Cycling conditions for Applied Biosystems Bio Rad Stratagene and Eppendorf cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15s DOE min 60 C Perform fluorescence data collection Recommended for the following cyclers Applied Biosystems models 5700 7000 7300 7500 7700 7900HT StepOnePlus ViiA 7 Bio Rad models iCycler iQ5 MyiQ MyiQ2 CFX96 CFX384 Stratagene models Mx3000P Mx3005P Mx4000P Eppendorf Mastercycler ep realplex models 2 2S 4 4S t For Bio Rad models CFX96 and CFX384 adjust the ramp rate to 1 C s For Eppendorf Mastercyler ep realplex models 2 2S 4 and 4S for the Silver Thermoblock adjust the ramp rate to 26 for the Aluminum Thermoblock adjust the ramp rate to 35 Refer to the Instrument Setup Guide at www SABiosciences com pcrarrayprotocolfiles php for detailed setup instructions Table 5 Cycling conditions for Roche cyclers Cycles Duration Temperat
3. Transcriptase Mix 28 ul Control P2 18 ul RNase Free Water 1 ml RT SYBR Green qPCR For 2 x 96 assays in 96 well plates 330500 Mastermix 2 suitable for use with real time cyclers that do not require a reference dye 2 x 1 25 ml Mastermix RT SYBR Green Fluor For 2 x 96 assays in 96 well plates 330510 qPCR Mastermix 2 suitable for use with real time cyclers that use fluorescein reference dye 2 x 1 25 ml Mastermix RT SYBR Green ROX For 2 x 96 assays in 96 well plates 330520 qPCR Mastermix 2 suitable for use with real time cyclers that use ROX reference dye 2 x 1 25 ml Mastermix RT SYBR Green ROX For 2 x 96 assays in 96 well plates 330620 FAST Mastermix 2 suitable for use with real time cyclers that use ROX reference dye including the Rotor Gene Q and Rotor Gene 6000 2 x 1 25 ml Mastermix Related products RT Profiler PCR Array Arrays of assays for disease pathway Varies or functionally related genes available in 96 well 384 well and Rotor Disc 100 format RT RNA QC PCR Array Array for quality control analysis prior Varies to experiments using RT Profiler PCR Arrays available in 96 well 384 well and Rotor Disc 100 formats Larger kit sizes available please inquire 26 RT qPCR Primer Assay Handbook 03 2011 Product Human XpressRef Universal Total RNA Mouse XpressRef Universal Total RNA Rat XpressRef Universal Total RNA RNeasy Mini Kit 50 RNeasy FFPE Kit 5
4. bands on a gel or dissociation peaks a Genomic DNA Use a no reverse transcription NRT control in contamination which reverse transcriptase is replaced with water in the cDNA synthesis reaction to detect DNA contamination If the difference in C values between the NRT control and a complete reaction for the same gene of interest is greater than 6 then any DNA contamination will not affect the reliability of the relative gene expression analysis We strongly recommend performing the on column DNase digestion step when purifying RNA using the RNeasy Mini Kit We strongly recommend using the RT First Strand Kit for cDNA synthesis This kit includes a genomic DNA elimination step b Presence of Approximately 4596 of all human genes are undiscovered predicted to have alternative transcripts yet alternative transcripts variants for only 996 of human genes have been annotated by the NCBI RT qPCR Primer Assays for genes with known variants amplify sequence common to all transcripts and detect the sum of their expression Primer design cannot account for genes with unannotated transcripts c Presence of primer Verify the presence of primer dimers by agarose dimers gel electrophoresis primer dimmers are 50 bp In size Use the appropriate RT SYBR Green Mastermix to prevent the appearance of primer dimers RT qPCR Primer Assay Handbook 03 2011 19 Comments and suggestions C values are too high gt 35 o
5. before beginning the protocol Be sure not to introduce bubbles into wells tubes when pipetting M Do not use DEPC treated water Use high quality nuclease free water M If precipitates are present in the Mastermix tubes warm the reagents at 42 C for 1 min and vortex briefly to dissolve Repeat if necessary M To ensure that each experimental sample yields a reliably detectable C value in real time PCR we recommend using undiluted cDNA template and a 1 10 dilution of cDNA template in separate reactions In addition prepare either duplicate or triplicate reactions for each template at each concentration M For every experimental sample prepare reactions for every gene of interest and for a single housekeeping gene or a set of housekeeping genes to normalize the raw data Choose housekeeping gene s known to not change their expression under the experimental conditions M Prepare a positive control reaction using template known to represent the genes of interest such as template generated from XpressRef Universal Total RNA MB To control for DNA contamination introduced during reaction setup prepare a no template control NTC reaction replacing template with water M To control for genomic DNA contamination perform one assay for each gene of interest and each housekeeping gene using an equivalent volume of product from the no reverse transcription NRT reaction performed for each RNA sample E Optional Generate a standard
6. using an RNA 6000 Nano LabChip Verify that there are sharp bands peaks present for both the 18S and 28S ribosomal RNAs Figure 1 Any smearing of the RNA bands or shoulders on the RNA peaks indicate that degradation has occurred in the RNA sample A B FU MDA231 285 un 8S e E NNW BUG 0 in o in al fo EM S 20 25 30 35 40 45 50 55 s Figure 1 Ribosomal RNA integrity M Agilent Bioanalyzer electropherogram of high quality total RNA showing sharp peaks for the 18S left and 285 right ribosomal RNA Due to high quality of the RNA peaks do not have shoulders especially to the left of each peak 3 Agarose gel electrophoresis shows sharp bands especially at the bottom of each band for 28S and 18S ribosomal RNA Genomic DNA contamination Eliminating genomic DNA contamination is essential for obtaining optimal real time gene expression results using RT qPCR Primer Assays Use of a no reverse transcription NRT control in which reverse transcriptase is replaced with water in the cDNA synthesis reaction is the most accurate way to detect DNA contamination If the difference in C values between the NRT control and a complete reaction for the same gene of interest is greater than 6 then any DNA When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety dat
7. your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com 4 RT qPCR Primer Assay Handbook 03 2011 Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding RT qPCR Primer Assays or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more informati
8. 0 PAXgene Blood RNA Kit 50 RNeasy Microarray Tissue Mini Kit 50 RNeasy Micro Kit 50 QlAamp RNA Blood Mini Kit 50 Contents 2 tubes each containing 100 ug human RNA at 1 mg ml 2 tubes each containing 100 ug mouse RNA at 1 mg ml 2 tubes each containing 100 ug rat RNA at 1 mg ml 50 RNeasy Mini Spin Columns Collection Tubes 1 5 ml and 2 ml RNase free reagents and buffers 50 RNeasy MinElute Spin Columns Collection Tubes Proteinase K RNase Free DNase DNase Booster Buffer RNase free buffers RNase Free Water 50 PAXgene Spin Columns 50 PAXgene Shredder Spin Columns Processing Tubes RNase Free DNase I RNase free reagents and buffers To be used in conjunction with PAXgene Blood RNA Tubes RNeasy Mini Spin Columns Collection Tubes QIAzol Lysis Reagent RNase free reagents and buffers 50 RNeasy MinElute Spin Columns Collection Tubes 1 5 ml and 2 ml RNase free DNase Carrier RNA RNase free reagents and buffers 50 QlAamp Mini Spin Columns 50 QlAshredder Spin Columns Collection Tubes 1 5 ml and 2 ml RNase free reagents and buffers Larger kit sizes available please inquire Cat no 338112 338114 338116 74104 73504 762174 73304 74004 52304 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requeste
9. 6 49 Experimental C GOl 19 17 C HKG 16 36 For each sample calculate the difference between the C values AC for each gene of interest and the housekeeping gene or the average C value of the set of housekeeping genes For example AC control C GOI C HKG 24 25 16 49 7 76 AC experimental Cr GOI C HKG 19 17 16 36 2 81 For each pair wise set of samples to be compared calculate the difference in AC values AAC for the genes of interest between the two samples For example AAC AC experimental AC control 2 81 7 76 4 95 Calculate the fold change in gene expression Due to high levels of amplification efficiency using RT qPCR Primer Assays the fold change in gene expression is equal to 214107 For example Fold change 244 204 75 2495 30 9 Standard curve method The standard curve method is an alternative method for data analysis Perform the standard curve method as described below Use the real time cycler software to determine the threshold cycle value for each reaction To generate a standard curve plot the threshold cycle Cr for each standard curve reaction y axis against the template dilution factor used in those reactions x axis log scale Plot a standard curve for each gene of interest GOI and for each housekeeping gene HKO Fit the data to an 22 RT qPCR Primer Assay Handbook 03 2011 equation defining a straight line The d
10. March 2011 RT qPCR Primer Assay Handbook For gene expression analysis by real time RT PCR QIAGEN Sample 8 Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Shipping and Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 4 Technical Assistance 5 Safety Information 5 Quality Control 5 Introduction 6 Principle and procedure 6 Description of protocols 6 Equipment and Reagents to Be Supplied by User 8 Important Notes 9 Preparing a workspace free of DNA contamination 9 RNA preparation quantification and quality control 9 Genomic DNA contamination 11 Starting RNA amounts 12 Protocols m cDNA Synthesis Using the RT First Strand Kit 13 B Real Time PCR Using RT qPCR Primer Assays and RT SYBR Green Mastermixes 15 Troubleshooting Guide 19 Appendix A Data Analysis 22 AAC method 22 Standard curve method 22 References 25 Ordering Info
11. The RT First Strand Kit includes a proprietary genomic DNA elimination step to remove any residual contamination in RNA samples before reverse transcription thereby eliminating false positive signals Each of the real time instrument specific RT SYBR Green Mastermixes contains SYBR Green and an appropriate reference dye to match the instrumentation available in your laboratory RT SYBR Green Mastermixes are available for all real time PCR instruments from QIAGEN Applied Biosystems Bio Rad Stratagene Eppendorf Roche and other major suppliers Description of protocols This handbook contains 2 protocols The first protocol details cDNA synthesis by reverse transcription using purified RNA and the RT First Strand Kit page 13 This protocol should be performed prior to real time PCR The second protocol 6 RT qPCR Primer Assay Handbook 03 2011 describes how to perform real time PCR using the cDNA prepared in the first protocol as template page 15 RT qPCR Primer Assay Handbook 03 2011 7 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier M RT First Strand Kit cat no 330401 M RT SYBR Green Mastermix suitable for use with your real time cycler RT SYBR Green Mastermixes available include m RT SYBR G
12. a sheets MSDSs available from the product supplier RT qPCR Primer Assay Handbook 03 2011 11 contamination will not affect the reliability of the relative gene expression analysis To remove any residual contamination from your RNA samples we strongly recommend RNA purification using the RNeasy Mini Kit including the optional on column DNase digestion step followed by cDNA synthesis using the RT First Strand Kit If required individual species specific RT qPCR Primer gDNA Controls are available Starting RNA amounts RT qPCR Primer Assays provide results with as little as 25 ng or as much as 5 ug total RNA per cDNA synthesis reaction For smaller starting RNA amounts the RT PreAMP cDNA Synthesis Kit cat no 330451 enables gene expression analysis from as little as 1 ng total RNA or 100 ng RNA from FFPE samples by preamplifying first strand cDNA This allows gene expression analysis from samples such as fine needle biopsy samples laser captured microdissection samples stem cell clusters or embryoid bodies FACS generated cells or FFPE samples For more details see the RT PreAMP cDNA Synthesis Handbook The optimal amount of starting material depends on the relative abundance of the transcripts of interest Lower abundance transcripts require more RNA higher abundance transcripts require less RNA Greater amounts of input total RNA yield a greater number of positive calls i e genes expressed in the linear dynamic rang
13. ay may be used solely in accordance with the RT qPCR Primer Assay Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the RT qPCR Primer Assay Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated ay CT SI The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com 2011 QIAGEN all rights reserved eee www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 T
14. curve for each gene of interest and housekeeping gene s To generate a standard curve prepare a 5 point series of 5 or 10 fold dilutions in duplicate using a template known to represent the genes of interest such as template generated from XpressRef Universal Total RNA RT qPCR Primer Assay Handbook 03 2011 15 Procedure 1 Briefly centrifuge the RT SYBR Green Mastermix RT qPCR Primer Assay and cDNA synthesis reaction 10 15 s to bring the contents to the bottom of the tubes Note As the RT SYBR Green Mastermix contains HotStart DNA Taq Polymerase that is active only after heat activation reactions can be prepared at room temperature 15 25 C 2 Prepare the PCR components mix in a 5 ml tube as described in Table 3 Table 3 PCR components mix for one reaction Component Volume RT SYBR Green Mastermix 12 5 ul cDNA synthesis reaction 1 ul RT qPCR Primer Assay 10 UM stock 1 ul RNase free water 10 5 ul Total volume 25 pl Note If performing multiple reactions prepare a mix containing RT SYBR Green Mastermix RT qPCR Primer Assay and RNase free water by scaling up the volumes shown in Table 3 Prepare 1096 more mix than is required to allow for pipetting errors i e for 96 reactions prepare enough PCR components mix for 106 reactions Add the mix to the cDNA synthesis reactions using a repeat pipet 3 Briefly centrifuge the PCR components mix and place the tube s into the real time cycler
15. d from QIAGEN Technical Services or your local distributor RT qPCR Primer Assay Handbook 03 2011 27 Notes 28 RT qPCR Primer Assay Handbook 03 2011 Notes RT qPCR Primer Assay Handbook 03 2011 29 Notes 30 RT qPCR Primer Assay Handbook 03 2011 Trademarks QIAGEN GllAzol RNeasy QlAamp Rotor Gene Rotor Disc QIAGEN Group PAXgene PreAnalytiX GmbH Roche LightCycler Roche Group Applied Biosystems ROX StepOnePlus ViiA Applera Corporation or its subsidiaries Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P Mx3000P Mx4000 Stratagene Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ Bio Rad Laboratories Inc FACS Becton Dickinson and Company DNA free Ambion Inc SYBR Molecular Probes Inc LabChip Caliper Technologies Corp Agilent Agilent Technologies Inc TRizol RNAzol Molecular Research Center Inc Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law LIMITED LICENSE STATEMENTS Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 994 056 and 6 171 785 The purchase of this product includes a limited nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own
16. e of the method Lower amounts of input total RNA yield a smaller number of positive calls For successful results we recommend that first time users start with 0 5 ug to 1 ug of total RNA It is important to use a consistent amount of total RNA for all reactions in a single experiment 12 RT qPCR Primer Assay Handbook 03 2011 Protocol cDNA Synthesis Using the RT First Strand Kit Important points before starting M Use the same amount of total RNA for reverse transcription of each sample to be analyzed First time users are recommended to start with 0 5 1 ug total RNA Use of less than 100 ng RNA will result in a high rate of false negatives MB Do not use DEPC treated water Use high quality nuclease free water M The RT First Stand Kit is not compatible with the chemicals in DNA free kits from Ambion If your RNA sample has been treated with DNA free reagents contact QIAGEN Technical Service Procedure 1 Briefly centrifuge the reagents of the RT First Stand Kit 10 15 s to bring the contents to the bottom of the tubes 2 Prepare the genomic DNA elimination mix for each RNA sample in a sterile PCR tube according to Table 1 Mix gently by pipetting up and down and then centrifuge briefly Table 1 Genomic DNA elimination mix Component Amount RNA 25 ng 5 ug Buffer GE 2 ul RNase free water Variable Total volume 10 ul 3 Incubate the genomic DNA elimination mix for 5 min at 42 C then place i
17. ease the efficiency of enzyme activities necessary for optimal reverse transcription and real time PCR performance Recommended RNA preparation methods High quality total RNA for your real time PCR experiment should be prepared using one of the methods described below depending on the biological sample For optimal results RNA samples should be suspended in RNase free water Do not use DEPC treated water Cultured cells We recommend the RNeasy Mini Kit cat no 74104 for RNA purification from cultured cells It is important to perform the on column DNase digestion step RT qPCR Primer Assay Handbook 03 2011 9 described in the RNeasy Mini Handbook using the RNase Free DNase Set cat no 79254 Tissue samples We recommend the RNeasy Microarray Tissue Mini Kit cat no 73304 including the optional on column DNase digestion step described in the RNeasy Microarray Tissue Handbook using the RNase Free DNase Set cat no 79254 Formalin fixed paraffin embedded FFPE samples We recommend the RNeasy FFPE Kit cat no 73504 for RNA purification from FFPE samples Small samples yielding 100 ng total RNA We recommend the RNeasy Micro Kit cat no 74004 for RNA purification from small samples Whole blood samples We recommend the PAXgene Blood RNA Kit cat no 762174 for preparation of total RNA from whole blood samples Alternatively the QlAamp RNA Blood Mini Kit cat no 52304 can also be used for thi
18. echnical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orde
19. ental sample and under the same PCR conditions specifically the initial template concentration whether undiluted or diluted 1 10 To determine the fold change in expression for each gene of interest between 2 different samples calculate the ratio of its normalized expression levels determined from the same PCR conditions between those samples For example Experimental L GOI 0 17 _ _Experimental L HKG 0 14 B oig change Control Gol ooa 7 Control L HKG 0 13 rr LL eee 24 RT qPCR Primer Assay Handbook 03 2011 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor i H E M s iat RT qPCR Primer Assay Handbook 03 2011 25 Ordering Information Product Contents Cat no RT qPCR Primer Assay For 200 reactions Mix of 2 gene Varies 200 specific primers provided in solution 200 ul 10 uM each primer RT First Strand Kit 12 For 12 x 20 ul first strand cDNA 330401 synthesis reactions Buffer GE 30 ul Buffer BC3 60 ul RE3 Reverse
20. es Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product of its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 The purchase of this product includes a limited non transferable license under specific claims of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology Inc and Roche Diagnostics GmbH to use only the enclosed amount of product according to the specified protocols No right is conveyed expressly by implication or by estoppel to use any instrument or system under any claim of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 other than for the amount of product contained herein Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the RT qPCR Primer Assay to the following terms 1 The RT qPCR Primer Ass
21. genes in first strand cDNA each potentially available as a PCR template In addition primers that provide efficient amplification are important to ensure accurate gene expression results from the commonly used AAC method which requires a consistently high degree of amplification efficiency across all experiments Taking advantage of an experimentally verified proprietary computer algorithm QIAGEN has developed high quality gene specific RT qPCR Primer Assays for gene expression analyses and microarray data validation RT qPCR Primer Assays are designed for SYBR Green based real time PCR detection The primer design computer algorithm has been developed using an in vitro assay to ensure that the resulting primer sequences generate a single PCR product of the predicted size and a minimal amount of primer dimer in 30 cycles of PCR amplification The assay also ensures that the amplification efficiency of the primers is at least 9096 As a result the algorithm designs highly effective primer sequences for SYBR Green based real time PCR detection RT qPCR Primer Assays are available for every human mouse rat rhesus macaque fruit fly and dog gene annotated by the NCBI Principle and procedure For optimal performance RT qPCR Primer Assays should be used together with the RT First Strand Kit for cDNA synthesis and RT SYBR Green Mastermixes for PCR These reagents have been formulated and pretested together with RT qPCR Primer Assays
22. ilution factor in the standard curve is directly related to the relative expression level L of its gene For example 30 25 20 15 10 Threshold Cycle 5 0 0 001 0 01 0 1 1 Dilution factor GOI C 3 302Log L 16 6 HKG C 3 351Log L 13 4 For every reaction containing template synthesized from experimental samples use the C value and the appropriate standard curve based on the gene specific RT qPCR Primer Assay used in the reaction to calculate the relative expression level of each gene of interest L GOI and the relative expression level of each housekeeping gene L HKG in each sample Be sure that the C values fall within the linear range of the appropriate standard curve For example No NN CO O O A O A O A O Threshold Cycle 001 0 01 0 1 1 Dilution Factor 0 0048 0 13 Control CAGOI 24 25 C HKG 16 49 L GOI 0 0048 L HKG 0 13 RT qPCR Primer Assay Handbook 03 2011 23 gt gt WY b CO O O A O A O A O Threshold Cycle 001 0 01 D 1 1 Dilution Factor 0 17 Experimental Cr GOI 19 17 C HKG 16 36 L GOI 0 17 L HKG 0 14 Normalize the expression level of the genes of interest by dividing their relative expression level by the relative expression level of the housekeeping gene or the average relative expression level of a set of housekeeping genes Be sure to use relative expression levels for all genes determined from the same experim
23. internal research No right under any other patent claim and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA This product is provided under an agreement between Molecular Probes Inc and SABiosciences and the manufacture use sale or import of this product is subject to one or more of U S Patent Nos 5 436 134 5 658 751 and corresponding international equivalents owned by Invitrogen Corp The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer where such research does not include testing analysis or screening services for any third party in return for compensation on a per test basis The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purpos
24. mmediately on ice for at least 1 min RT qPCR Primer Assay Handbook 03 2011 13 4 Prepare the reverse transcription mix according to Table 2 Table 2 Reverse transcription mix Volume Volume Volume for 1 for 2 for 4 Component reaction reactions reactions 5x Buffer BC3 4 ul 8 ul 16 ul Control P2 1 ul 2 ul 4 ul RE3 Reverse Transcriptase Mix 2 ul 4 ul 8 ul RNase free water 3 ul 6 ul 12 ul Total volume 10 ul 20 ul 40 pl 5 Add 10 ul reverse transcription mix to each tube containing genomic DNA elimination mix Mix gently by pipetting up and down 6 Incubate at 42 C for exactly 15 min Then immediately stop the reaction by incubating at 95 C for 5 min 7 Add 91 ul RNase free water to each reaction Mix by pipetting up and down several times 8 Place the reactions on ice and proceed with the real time PCR protocol If you wish to store the reactions prior to real time PCR transfer them to a 20 C freezer For quality control analysis using the RT RNA QC PCR Array follow the protocol in the RT RNA QC PCR Array Handbook using a 6 ul aliquot of the diluted cDNA template oo 14 RT qPCR Primer Assay Handbook 03 2011 Protocol Real Time PCR Using RT qPCR Primer Assays and RT SYBR Green Mastermixes Important points before starting M Ensure that the RT SYBR Green Mastermix is suitable for your real time cycler see page 8 EM For accuracy and precision ensure that micropipettors are calibrated
25. on please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s Quality Management System each lot of RT qPCR Primer Assays is tested against predetermined specifications to ensure consistent product quality RT qPCR Primer Assay Handbook 03 2011 5 Introduction Real time RT PCR is a highly sensitive and reliable method for gene expression analysis for multiple applications such as the verification of microarray data Optimal primer design is critical for successful real time PCR based analysis of gene expression Carefully designed primers specifically amplify genes of interest overcoming the challenge of eliminating nonspecific amplification due to the presence of thousands of
26. on of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed gene expression profiles and false positive signals The most common sources of DNA contamination are the products of previous experiments spread into the air of the working environment To set up and maintain a working environment free of DNA contamination follow the guidelines below M Wear gloves throughout the procedure Use only fresh PCR grade reagents water and labware tips and tubes M Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate the PCR workspace and labware pipettor barrels tube racks etc before each use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 1096 bleach to chemically inactivate and degrade any DNA M Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any labware tips or tubes containing PCR products or other DNA treat with 1096 bleach M Do not leave labware tubes and tip boxes exposed to the air for long periods of time RNA preparation quantification and quality control The most important prerequisite for any gene expression analysis experiment is consistent high quality RNA from every experimental sample Residual traces of proteins salts or other contaminants may degrade the RNA or decr
27. r not detectable a Experimental error b Poor RNA quality c Insufficient template d Nonendogenous transcript Use a template known to contain the gene of interest as a positive control to check the PCR reagents and experimental procedure Be sure to perform all recommended quality control checks on the RNA sample Poor quality RNA can inhibit enzyme activity during reverse transcription generating an insufficient amount of template during the cDNA synthesis reaction Use more input RNA for reverse transcription especially if the lower end of the recommended range had been used previously Use a larger volume of template per reaction but do not use more than 2 5 ul of template per 25 ul reaction Use the same volume of template in each reaction High or undetectable C values will result if the target gene is exogenously expressed from a vector plasmid or other construct that only contains the open reading frame and the RT qPCR Primer Assay is located in the 3 or 5 untranslated region UTR Refer to the reference positions on the Product Sheet provided with the RT qPCR Primer Assay C values are too low 12 Too much template Use less input RNA for cDNA synthesis especially if the higher end of the recommended range had been used previously Use a smaller volume of template per reaction but do not use less than 1 ul of template per 25 ul reaction Use the same volume of template in each reaction
28. reen qPCR Mastermix suitable for use with real time cyclers that do not require a reference dye including Bio Rad models CFX96 CFX384 Bio Rad MJ Research models Chromo4 DNA Engine Opticon 2 Roche LightCycler 480 96 well and 384 well m RT SYBR Green Fluor qPCR Mastermix suitable for use with the following real time cyclers Bio Rad models iCycler iQ 5 MyiQ MyiQ2 m RT SYBR Green ROX gPCR Mastermix suitable for use with the following real time cyclers Applied Biosystems models 5700 7000 7300 7500 Standard and Fast 7700 7900HT Standard and Fast 96 well block 384 well block StepOnePlus ViiA 7 Standard and Fast 96 well block 384 well block Eppendorf Mastercycler ep realplex models 2 2S 4 4S Stratagene models Mx3000P Mx3005P Mx40005 Takara TP 800 m RT SYBR Green ROX FAST Mastermix suitable for use with the Rotor Gene Q and other Rotor Gene cyclers Purified RNA samples Real time PCR cycler High quality nuclease free water Do not use DEPC treated water Nuclease free pipet tips and tubes Optional XpressRef Universal Total RNA to control PCR conditions is available for human cat no 338112 mouse cat no 338114 and rat cat no 338116 E AAE KK el 8 RT qPCR Primer Assay Handbook 03 2011 Important Notes Preparing a workspace free of DNA contamination For accurate and reproducible PCR array results it is important to avoid contaminati
29. rmation 26 RT qPCR Primer Assay Handbook 03 2011 3 Kit Contents RT qPCR Primer Assay 200 Catalog no 330001 Number of 25 pl reactions 200 200 ul RT qPCR Primer Assay 10 UM in a single tube tube Shipping and Storage The RT qPCR Primer Assays are shipped at ambient temperature but must be stored at 20 C upon arrival When stored under these conditions and handled correctly the product can be kept for at least 6 months from date of receipt without reduction in performance Product Use Limitations RT qPCR Primer Assays are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet
30. rs 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 e Q e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 106 DE USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN dc Sample 8 Assay Technologies
31. s purpose Total RNA isolated using a phenol based method Total RNA from any biological source material prepared using a phenol based method e g GlAzol Lysis Reagent TRIzol Reagent RNAzol Reagent should be further purified using the RNeasy Mini Kit It is important to perform the on column DNase digestion step described in the RNeasy Mini Handbook Other biological samples Refer to the existing literature to find protocols for high quality RNA purification from other biological samples or contact QIAGEN Technical Service RNA quantification and quality control For best results from the RT qPCR Primer Assays all RNA samples should also demonstrate consistent quality according to the following criteria Concentration and purity determined by UV spectrophotometry The concentration and purity of RNA should be determined by measuring the absorbance in a spectrophotometer Prepare dilutions and measure absorbance 10 RT qPCR Primer Assay Handbook 03 2011 in 10 mM Tris Cl pH 8 0 The spectral properties of nucleic acids are highly dependent on pH An absorbance reading of 1 0 at 260 nm in a 1 cm detection path corresponds to an RNA concentration of 40 ug ml EN AosoiAsag ratio should be greater than 1 7 i Ax60 A280 ratio should be 8 to 2 0 Hi Concentration determined by A should be gt 40 ug ml Ribosomal RNA band integrity Run an aliquot of each RNA sample on a denaturing agarose gel or the Agilent Bioanalyzer
32. ure Comments 1 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 45 15s 95 C min 60 C Perform fluorescence data collection Recommended for the Roche LightCycler 480 If using a Roche LightCycler 480 adjust the ramp rate to 1 C s Refer to the Instrument Setup Guide at www SABiosciences com pcrarrayprotocolfiles php for more information on other required changes to settings for Melt Curve Acquisition oo AA AA RT qPCR Primer Assay Handbook 03 2011 17 Table 6 Cycling conditions for Bio Rad and Takara cyclers and all other cyclers Cycles Duration Temperature Comments 1 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15s 95 C 30 40 s 55 C Perform fluorescence data collection Different cyclers need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 55 C for your cycler 30s 12 Recommended for the following cyclers Bio Rad MJ Research models Chromo4 DNA Engine Opticon DNA Engine Opticon 2 Takara TP 800 all other cyclers 5 Recommended Perform dissociation melting curve analysis to verify PCR specificity Run a melting curve program and generate a first derivative dissociation curve for each well using the real time cycler software A single peak should appear in each reaction at temperatures greater than 80 C Note If your instrument does not have a default melting c
33. urve program run the following program instead 95 C 1 min 65 C 2 min optics off 65 C to 95 C at 2 C min optics on Note For cycler specific melting curve analysis settings please refer to the Instrument Setup Guide for your cycler at www SABiosciences com pcrarrayprotocolfiles php Note Reactions can be stored at 20 C wrapped in aluminum foil and melting curve analysis performed at a later time When ready to perform melting curve analysis warm the tube to room temperature 15 25 C place it in the real time cycler and run the melting curve analysis program 6 Optional Agarose gel electrophoresis analysis can be performed if necessary for troubleshooting purposes No more than one band should be visible in each lane The RT qPCR Primer Assay Product Sheet details the expected size of the PCR product 18 RT qPCR Primer Assay Handbook 03 2011 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www SABiosciences com support faq php target PCR The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Presence of multiple PCR products
Download Pdf Manuals
Related Search