User Manual - SABiosciences
Contents
1. 11 Ill Additional Materials Required A Genomic DNA Isolation See Page 14 for specific recommendations B High quality nuclease free H2O DO NOT USE DEPC H20 C Equipment 1 For recommendations on specific real time instrumentation thermal cyclers with fluorescent detection see the list of plate formats above a NOTE The qBiomarker Somatic Mutation PCR Arrays are NOT recommended for the Cepheid SmartCycler the Roche LightCycler 2 0 or the QIAGEN Rotor Gene Q due to the different non traditional hot block arrangements in these instruments 2 Calibrated Multi Channel Pipettor 3 RNase DNase free pipette tips and tubes i Optional A QIAGEN REPLI g UltraFast Mini Kit Cat 150033 12 qBiomarker Somatic Mutation PCR System IV Protocol Please read through this entire protocol before beginning your experiment The chemically modified and tightly controlled HotStart enzyme along with other proprietary chemical components in the qBiomarker Probe Mastermixes uniquely provide accurate hydrolysis probe assay PCR results by preventing the amplification of non specific products The combination of these reagents also helps ensure high amplification efficiencies for all the assays Because each instrument uses a different reference dye to normalize its optics be sure that you use the correct master mix for the instrumentation in your laboratory NOTE Preparing a Workspace Free of DNA Contamination Fo2. BILo E Ua A zz 5 MOO oz 7o O x r n m e o te 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Sample 4 112 3 4 5 6 7 28 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 7o O r7 A zz 5 MOO oz Be GE a 0 aa Laal Gea gt 1 2 3 4 5 6 7 8 9 10 11 22 13 14 15 16 17 18 19 20 21 22 23 24 Figure 4 To load a 384 well format qBiomarker Somatic Mutation PCR Array add 10 ul of the Experimental Cocktail from each numbered sample into the staggered wells with the same number as indicated in the figure 20 qBiomarker Somatic Mutation PCR System i Proceed to the next section STEP 3 on Performing Real Time PCR Detection c Performing Real time PCR Detection NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument i CAREFULLY but tightly seal the qBiomarker Somatic Mutation PCR Array with the optical thin wall 8 cap strips Formats A and D or with the optical adhesive film Formats C E F and G NOTE Be sure that no bubbles remain in any of the wells of the qBiomarker Somatic Mutation PCR Array To remove bubbles tap the plate gently on the bench top and centrifuge the plate at 1000 rpm for 1 minute for 96 well plate or at 2000 rpm for 2 minutes for 384 well plate
3. ii Place the plate on ice while setting up the PCR cycling program below iii Place one plate in your real time thermal cycler Use a compression pad with the optical film sealed plate formats Formats C E F and C if recommended by your instrument s user manual NOTE qBiomarker Somatic Mutation PCR Arrays containing experimental cocktail that will not be processed immediately may be stored wrapped in aluminum foil at 20 C for up to one week until ready to run iv Enter and run the appropriate program for your real time instrument Cycles Duration Temperature 1 10 minutes 95 C 15 seconds 95 C 40 1 minute 60 C 1 The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record FAM fluorescence from every well during the annealing step of each cycle 21 d Calculate the threshold cycle C for each well using the instrument s software We highly recommend manually setting the Baseline and Threshold Values To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number five 5 through two 2 cycle values before the earliest visible amplification usually around cycle number fifteen 15 but no more than twenty 20 To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lower half to one third of the linear phase of the amplifi
4. should be resuspended in DNase free water or alternatively in DNase free 10 mM Tris buffer pH 8 0 DO NOT use DEPC treated water 2 DNA Quality Control For best results from the qBiomarker Somatic Mutation PCR Array all DNA samples should also demonstrate consistent quality according to the following criteria a DNA Concentration and Purity by UV Spectrophotometry NOTE Prepare dilutions and measure absorbance in 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH i Concentration by Azgo should be greater than 10 ug ml DNA ii A260 A2so ratio should be greater than 1 8 iil A260 A23o ratio should be greater than 1 7 b DNA Integrity In order to start with 10 ng genomic DNA with the whole genome amplification and achieve the best PCR array results the genomic DNA should be greater than 2kb in length with some fragments greater than 10kb This can be checked by running a fraction of each DNA sample on a 196 agarose gel For DNA extracted 14 qBiomarker Somatic Mutation PCR System from FFPE sections we recommend skipping the amplification process c DNA QC Plate DNA quality and consistency can also be checked on the qBiomarker Somatic Mutation PCR Array Human DNA QC Plate cat no 337021 SMH 999AFA by measuring 7 reference genes in real time PCR Please refer to Appendix A for details NOTE When uncertain of your samples quality by the above methods please contac
5. 100 Each qBiomarker Somatic Mutation PCR Array shipment includes the arrays and either 12 optical thin wall 8 cap strips Formats A and D or one optical adhesive film Formats C E F and G per array Each 96x4 Format 384 Well qBiomarker Somatic Mutation PCR Array Formats E and G also includes one set of 4 384EZLoad Covers Catalog 338125 for each qBiomarker Somatic Mutation PCR Array provided in the package NOTE Each 384EZLoad Cover is for a Single Use ONLY 2 qBiomarker Probe Mastermixes Be sure to pick the correct one for the instrumentation in your laboratory qBiomarker Probe Mastermix ROX Specifically designed for e All ABI and Stratagene Instrumentation e All Instruments that do not require a reference dye such as o Bio Rad Opticon Opticon 2 and Chromo 4 o Roche LightCycler 480 System o Eppendorf Mastercycler ep realplex 2 2S 4 4S qBiomarker Probe Mastermix Fluorescein Specifically designed for e BioRad iCycler MyiQ and iQ5 Instrumentation 10 qBiomarker Somatic Mutation PCR System Shipping amp Storage Conditions Please check the kit components immediately after you receive this package We are only responsible for missing items reported within two 2 business days of receipt qBiomarker Somatic Mutation PCR Arrays 337021 SMX V F R Y ZZ e X Species H Human THHE Somatic Mutation Pathway Identifier V Design Version Y Plate Format ZZ Pack Size F R Accompanying
6. 22 12 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN e E Sample amp Assay Technologies UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999
7. 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 1122 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 Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254
8. Master Mix with Reference Dye Fluorescein or ROX Shipping Conditions qBiomarker Somatic Mutation PCR Arrays are shipped at Room Temperature RT or on Blue Ice BI Storage Conditions Keep plates at 20 C for long term storage qBiomarker Probe Mastermixes Shipping Conditions qBiomarker Probe Mastermixes are shipped on Blue Ice BI Storage Conditions Keep qBiomarker Probe Mastermixes at 4 C for long term storage NOTE Be sure that you have the correct qBiomarker Somatic Mutation PCR Array format and Mastermix with correct Reference Dye for your instrument before starting the experiment qBiomarker Somatic Mutation PCR Assays 337011 SMPX IHHHHHEA R F e X Species H Human e THHHHHIL Somatic Mutation Identification Code e A Design Version e R F Accompanying Master Mix with Reference Dye ROX or Fluorescein Shipping Conditions qBiomarker Somatic Mutation PCR Assays are shipped on Blue Ice BI Storage Conditions Keep qBiomarker Somatic Mutation PCR Assays at 20 C for long term storage qBiomarker Probe Mastermixes Shipping Conditions qBiomarker Probe Mastermixes are shipped on Blue Ice BI Storage Conditions Keep qBiomarker Probe Mastermixes at 4 C for long term storage NOTE Be sure that you have the correct Mastermix with correct Reference Dye for your instrument before starting the experiment When stored properly at the recommended conditions their performance is guaranteed for 6 months
9. St 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 NOTICE TO PURCHASERS Use of kit components for reproduction of any primer pair mix to modify kit components for resale or to use qBiomarker Somatic Mutation PCR Arrays amp Assays to manufacture commercial products without written approval of SABiosciences Corporation is expressly prohibited PRODUCT WARRANTY This warranty limits our liability to the replacement of this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product LIMITED LICENSE STATEMENTS Use of this product is covered by one or more of the following US patents and corresponding patent cla
10. and qBiomarker Probe Mastermixes have been pre optimized hand in hand for hydrolysis probe based real time RT PCR detection The simplicity of the qBiomarker Somatic Mutation PCR Array format and operating procedure allows routine somatic mutation profiling in any research laboratory with access to real time PCR instruments Benefits of the qBiomarker Somatic Mutation PCR Arrays Pathway Focused Profile the somatic mutation status of important genes relevant to major signal transduction pathways Simple and Accurate Simple real time PCR procedure provides high sensitivity and wide dynamic range Designed for Routine Use Bring somatic mutation profiling to any lab with a real time PCR instrument Figure 2 Overview of the qBiomarker Somatic Mutation PCR Array Assay Protocol Extraction Amplification Detection Analysis DNA mini or REPLI g for Xx qPCR Array FFPE kit fresh sample and Assay ps 30 min 90 min 2 hrs 10 20 min Figure 2 Overview of the qBiomarker Somatic Mutation PCR Array Assay Protocol The procedure involves DNA extraction QIAGEN QlAamp DNA Mini Kit or QlAamp DNA FFPE Tissue Kit are recommended an optional amplification QIAGEN REPLI g Kit or REPLI g UltraFast Mini Kit are recommended step for DNA isolated from fresh samples qPCR detection on qBiomarker Somatic Mutation PCR Arrays or Assays and data analysis using the qBiomarker Somatic Mutation Data Analysis Template An optional DNA sample QC step
11. following address http www SABiosciences com SomaticMutationAnalysis php i Click on the qBiomarker Somatic Mutation PCR Array Data Analysis Template link ii Save the Excel file to your local computer Open the file in Excel iii Follow the instructions for using the template provided in the Instructions worksheet iv If using a 384 well format E or G similarly download the 384 Well Format E Data Analysis Patch to convert a 384 well dataset into the correct four sets of 96 assays for each of the four samples 2 Principles for qBiomarker Somatic Mutation PCR Data Analysis The qBiomarker Somatic Mutation PCR Assay utilizes allele specific primer design Each mutation assay maximizes the detection of mutant DNA with minimal or no detection of the wild type DNA template The Ct value CtVU from the mutation specific assay is inversely correlated to the abundance of mutant DNA in the sample i AACt method recommended for experiments using fresh unfrozen or frozen samples or smaller lt 4 number of samples To account for the different amounts of starting DNA copies used in the experiment a separate reference assay is setup using the same amount of DNA as used in the mutation specific assay This reference assay is designed on a non variable region of the same gene which carries the mutation and its Ct value Ct essentially correlates to the total copies of DNA used in the mutation specific assay NOTE Hi
12. immediately before the detection array or assay setup allows the user to qualify the DNA samples For DNA QC Plate Setup refer to Appendix A on page 27 qBiomarker Somatic Mutation PCR System 7 Human EGFR Somatic Mutation PCR Array 123 10 11 12 A al pl 62 van B ove yn yu vis C vi6 v17 v18 95 56 67 D 68 69 610 aaa E e4 sd aou Fee a2 n3 nt Gere e 4 H 6 at pu t SPC SPC Somatic Copy Positive PCR Mutations Number Controls Figure 3 Layout of Pathway Focused qBiomarker Somatic Mutation PCR Arrays Wells A1 through H1 contain assays for somatic mutations in the same biological pathway Wells H2 through H10 contain gene copy control assays to normalize PCR Array data Depending on the specific array content slight variations in plate layout can occur Wells H10 through H12 contain replicate Positive PCR Controls SMPC to test for the presence of inhibitors in the sample or efficiency of the polymerase chain reaction itself using a pre dispensed artificial DNA sequence and the primer set that detects it labeled 1 4 in gray above contains the same primer set represented by the 96 well The 384 well format of the qBiomarker Somatic Mutation PCR Arrays includes four designations replicates of the same 96 well format in which each two by two set of wells wells 96 well qBiomarker Somatic Mutation PCR System Il Materials Provided 1 qBiomarker Somatic Mutation PCR Array Plate
13. 0 Stratagene Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ Bio Rad Laboratories Inc ROX StepOnePlus ViiA Applera Corporation or its subsidiaries SmartCycler Cepheid Excel Microsoft Corporation ARMS AstraZeneca 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 Agreement Use of this product signifies the agreement of any purchaser or user of the qBiomarker Somatic Mutation PCR Array or Assay to the following terms 1 The qBiomarker Somatic Mutation PCR Array and qBiomarker Somatic Mutation PCR Assay may be used solely in accordance with the qBiomarker Somatic Mutation 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 qBiomarker Somatic Mutation 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 ON eee
14. March 2011 qBiomarker Somatic Mutation PCR Handbook qBiomarker Somatic Mutation PCR Array qBiomarker Somatic Mutation PCR Assay For real time PCR based pathway focused somatic mutation profiling QIAGEN Sample amp 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 E Purification of DNA RNA and proteins M Nucleic acid and protein assays M microRNA research and RNAi E Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Product Use Limitations The gBiomarker Somatic Mutation PCR Array and qBiomarker Somatic Mutation PCR Assay products 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 CONTENTS Background and Introduction Il Materials Provided III Additional Materials Required IV Proto
15. and verifying drug target biomarkers for targeted therapy research involving the EGFR signaling pathway and downstream effectors For targeted therapy research studying the most common and clinically relevant mutations in the context of biological pathways provides more coverage and thus the most potential for the discovery and verification of clinical biomarkers Principle and Procedure Real time PCR is the most sensitive and reliable method for the detection of DNA mutations By combining allele specific amplification and hydrolysis probe detection real time PCR assays have been developed which can detect as low as 1 somatic mutations in the background of wild type genomic DNA Allele specific amplification is achieved by Amplification Refractory Mutation System ARMS technology which is based on the discrimination by Taq polymerase between a match and a mismatch at the 3 end of the PCR primer Figure 1 qBiomarker Somatic Mutation PCR System Mutant template Additional mismatch ARMS primer Mutation of interest Taq ARMS primer extends on mutant target DNA Wildtype template Q d ARMS primer n 3 LILILLLPRBLPLLILLLLLPRLLLLPLLLLILILII ARMS primer does not extend on wild type DNA Figure 1 Amplification Refractory Mutation System ARMS The qBiomarker Somatic Mutation PCR Arrays are designed to analyze a panel of somatic mutations reported in the important genes related to a biological pathway The mutations are select
16. are mixed well after thawing a Setting Up Real Time PCR Reaction Set up the following two reactions for the detection of one somatic mutation Do not forget to include a wildtype control sample i Specific somatic mutation assay ii Corresponding reference gene copy assay Table 6 Individual PCR Assay Setup qBiomarker Probe 12 5 ul Mastermix Mutation PCR Assay NOTE t is recommended to prepare a cocktail containing qBiomarker Probe Mastermix genomic DNA and H20 for the total number of PCR reactions Then aliquot this cocktail into different PCR wells with pre dispensed primer assays b Performing Real time PCR Detection NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument i CAREFULLY but tightly seal the PCR plate with the optical thin wall 8 cap strips or with the optical adhesive film NOTE Be sure that no bubbles remain in any of the wells To remove bubbles tap the plate gently on the bench top and centrifuge the plate at 1000 rpm for 1 minute for the 96 well plate ii Place the plate on ice while setting up the PCR cycling program described below 23 Place one plate in your real time thermal cycler Use a compression pad with the optical film sealed plate formats if recommended by your instrument s user manual iv Enter and run the appropriate program for your real time instrument 7 1 The 10 minute step at 95 C
17. ate In order to determine the quality of DNA samples based on the Ct results first make sure that the Ct of SMPC assay for each sample is consistent at 22 If not please adjust the baseline and threshold setting to achieve that value Then calculate the average for the lowest 6 Cts among the gene copy number assays for each sample The highest Ct is removed from the average calculation as some samples may contain homozygous deletion for one of the 7 genes included on the QC plate The deleted gene will give a high Ct value The typical average Ct for high quality DNA from fresh tissue samples should be below 29 based on the Baseline and Threshold setup outlined on page 16 Samples of lower quality i e average Ct value higher than 29 may not yield optimal results For DNA extracted from FFPE samples an average Ct value of lower than 32 for the lowest 6 Cts using 2 ng gDNA input in 10 ul reaction volume or 5 ng gDNA in 25 ul reaction indicates sufficient quality for mutation profiling analysis Samples of lower quality i e average Ct value higher than 32 may not yield optimal results or require more input materials to make the average Ct value lower than 32 28 qBiomarker Somatic Mutation PCR System V Troubleshooting and FAQs 1 Evidence of Poor PCR Amplification Efficiency The average C S value varies by more than two 2 across the qBiomarker Somatic Mutation PCR Arrays being compared and or is greater than 24 Diff
18. cation plot The following settings serve as references for a few selected real time PCR instruments ABI 7900HT baseline setting 5 18 cycles threshold setting 0 1 ABI 7500 baseline setting 3 15 cycles threshold setting 0 1 Stratagene Mx3000p Mx3005p baseline setting adaptive threshold setting 0 1 IMPORTANT Ensure that the Baseline and Threshold are the same across all PCR array runs in the same analysis If the DNA sample quality has been adequately controlled the cycling program has been executed properly and the Baseline and Threshold have been defined correctly then the value of C should be 22 2 across all of your arrays or samples If not see the Troubleshooting and FAQ section Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with our Data Analysis Template Excel file 22 qBiomarker Somatic Mutation PCR System C Somatic Mutation PCR Assay Protocol NOTE To detect single somatic mutation using individual PCR assay there is no need to use amplified genomic DNA We recommend using 5 ng genomic DNA per PCR assay for DNA extracted from fresh tissue samples at least 5 ng and up to 30 ng genomic DNA per PCR assay for DNA of FFPE sample origin NOTE Be sure to use the correct master mix for your instrument before continuing with this protocol NOTE Thaw genomic DNA sample and qBiomarker Probe Mastermix at room temperature 15 25 C Make sure they
19. col A Genomic DNA Preparation and Quality Control B qBiomarker Somatic Mutation PCR Array Protocol C qBiomarker Somatic Mutation PCR Assay Protocol D Data Analysis E DNA QC Plate V Troubleshooting and Frequently Asked Questions 12 13 14 16 23 25 27 29 Introduction Acquisition of somatic mutations in human genomic DNA gDNA is an important event during tumorigenesis and cancer progression Somatic mutations occur as single mutations within a gene multiple mutations within a gene or mutations present across related genes in a variety of cancers Cells may respond differently to treatment regimens based on their somatic mutation profile For example the mutation status of the EGFR and KRAS genes can predict the physiological response to certain drugs targeting these molecules The utility of individual and multiple somatic mutation status information in identifying key signaling transduction disruptions has been demonstrated in numerous research studies The pathway focused qBiomarker Somatic Mutation PCR Arrays are translational research tools that allow rapid and accurate profiling of the somatic mutation status for a pathway focused set of genes and key downstream and associated signaling genes For example the EGFR Pathway qBiomarker Somatic Mutation PCR Array with its comprehensive content coverage is designed for studying mutations in the context of the EGFR pathway and has the potential for discovering
20. control samples The average Ct method assumes that for a given locus mutation only occurs in a small percentage of tested samples Thus the average Ct for that locus across all the samples analyzed can be used to represent the mutation assay background in the wild type sample The Ct from a mutation assay in a test sample will be compared with this average Ct If a particular mutation assay in a test sample yields a much lower Ct according to a present threshold than the average Ct for the same locus then this suggests that the sample carries a mutation at that locus Limited by the accuracy of the real time PCR chemistry any sample with a Ct value greater than 35 for the mutation specific assay indicates that the mutation is not detected for the corresponding allele in that sample A small number of assays will have a raw Ct cutoff of 36 or 37 These Ct cutoff values will be embedded in the qBiomarker Somatic Mutation PCR Array data analysis template 26 qBiomarker Somatic Mutation PCR System E Appendix A DNA QC Plate Setup Sample DNA quality can affect the performance of the somatic mutation PCR array For DNA extracted from FFPE sections different degrees of cross linkage and fragmentation may cause the mutation detection window to decrease consequently the mutation analysis for certain low quality samples may be compromised especially for mutant alleles that are present at a lower percentage in the sample Thus when not certa
21. ed from comprehensive somatic mutation databases e g COSMIC and peer reviewed scientific literature based on their clinical or functional relevance and frequency of occurrence To complete the qBiomarker Somatic Mutation PCR Array procedure Figure 2 start with 5 to 10 ng genomic DNA isolated from fresh unfrozen or frozen human tissues or as low as 200 ng DNA from formalin fixed paraffin embedded FFPE sections DNA from fresh tissues can be uniformly amplified using QIAGEN REPLI g UltraFast Mini Kit Then mix your DNA with the included ready to use qBiomarker Probe Mastermixes and aliquot the mixture into each well of the same plate containing pre dispensed gene specific primer and hydrolysis probe sets By performing real time PCR the mutation status of a particular sample is determined by comparing the allele specific Ct values between your test sample and a wild type control sample see the qBiomarker Somatic Mutation Data Analysis section for detailed principles Each array contains a panel of hydrolysis probe assays for a stringently selected set of pathway focused somatic mutations gene copy number controls and PCR quality controls The qBiomarker Somatic Mutation PCR Arrays are available in both 96 well and 384 well plate formats containing either one or four replicates respectively of the 96 assay set panel see Figure 3 for the layout of a typical qBiomarker Somatic Mutation PCR Array qBiomarker Somatic Mutation PCR Assays
22. erent instruments have different levels of sensitivity If an average C value of 22 2 is difficult to obtain for your instrument the observed average C S value should be acceptable as long as it does not vary by more than two cycles between qBiomarker Somatic Mutation PCR Arrays being compared Be sure that the initial heat activation step at 95 C has been lengthened to 10 minutes from the shorter time in the default program Be sure that all other cycle parameters also have been correctly entered according to the recommendations in this handbook If you have additional questions please check our website www SABiosciences com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 1 888 503 3187 or 301 682 9200 29 Ordering Information Product Contents Cat no qBiomarker Somatic PCR plate and master mix Varies Mutation PCR Array qBiomarker Somatic PCR assay and master mix Varies Mutation PCR Assay 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 requested from QIAGEN Technical Services or your local distributor Trademarks QIAGEN REPLI g QlAamp Rotor Gene QIAGEN Group Roche LightCycler Roche Group Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P Mx3000P Mx400
23. gher than normal Ct value means that the starting DNA amount and or quality is significantly lower than the optimal condition and this will reduce the ability to detect 1 mutant DNA in the sample For 5ng gDNA isolated from fresh tissue CtFF value typically ranges from 25 to 29 depending on target genes However if only one ctf shows aberrantly high value i e gt 35 while CtFF values for other genes are in the normal range this may indicate that a homologous deletion has happened for that gene None of the loci for a deleted gene will be assigned a genotype in downstream analysis The relative abundance of mutant DNA templates in a given test sample can be represented by ACtrest CtV CtREF 25 In order to reliably determine the mutation status for a specific allele in the test sample a control sample which has the wild type sequence for the corresponding allele also needs to be tested with the same mutation specific assay and reference assay The resulting ACtctri CtVUT CtFF establishes the wild type background relative to the total DNA input for the mutation specific assay When ACtrzsr is significantly smaller than ACtctri ACtrgsr lt ACtctr by statistical analysis or a preset threshold a positive mutation call can be made Otherwise the sample is considered to be wild type for the assayed allele ii Average Ct method recommended for experiments using FPPE samples large number of samples or without wild type
24. ims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 5 677 152 claims 1 to 23 only 5 773 258 claims 1 and 6 only 5 407 800 5 322 770 5 310 652 5 210 015 5 487 972 5 804 375 5 538 848 5 723 591 5 876 930 6 030 787 6 258 569 6 214 979 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims in US Patent No 6 814 934 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 from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA 2011 QIAGEN all rights reserved EE LL SSS aa cs msg 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 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax
25. in about the sample quality it is recommended to check the DNA quality first using a qBiomarker Somatic Mutation PCR Array Human DNA QC Plate cat no 337021 SMH 999AFA The DNA QC Plate is designed to measure the Ct of 7 reference genes When the DNA is highly cross linked or fragmented the Cts from these 7 genes will be much higher than those from the same amount of high quality DNA Each QC plate is enough for 12 DNA samples with the following 96 well layout each column is for one sample fea ee I aa I a 8IXERDESIERIDS B kras kras kras keas Kras Kras kras kras kras Kras Kras KRAS HRAS HRAS HRAS HRAS HRAS HRAS HRAS HRAS HRAS HRAS D NRAS Neas NRAS NRAS NRAS NRAS NRAS NRAS NRAS NRAS NRAS NRAS 1 Setting up the DNA QC Plates a 96 Well Format i Prepare the following reaction mix for each sample enough for 8 4 reactions reaction 8 4 reactions DNA Sample 40ng Probe Mastermix ii For each sample add 25 ul reaction mix to each assay well in the same column b 384 Well Format i Prepare the following reaction mix for each sample enough for 8 4 reactions 27 Ct reaction 8 4 reactions DNA Sample qBiomarker 5 ul 42 ul onmes Mastermix HO 10 pl 84 yl ii For each sample add 10 ul reaction mix to each assay well 2 Perform real time PCR run Please refer to Section IV B 2 d for detailed instructions 3 Data analysis of the QC pl
26. is required to activate the HotStart DNA polymerase Detect and record FAM fluorescence from every well during the annealing step of each cycle c Calculate the threshold cycle C for each well using the instrument s software We highly recommend manually setting the Baseline and Threshold Values ii To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number five 5 through two 2 cycle values before the earliest visible amplification usually around cycle fifteen 15 and twenty 20 To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lower half to one third of the linear phase of the amplification plot The following settings serve as references for a few selected real time PCR machines ABI 7900HT baseline setting 5 18 cycles threshold setting 0 1 ABI 7500 baseline setting 3 15 cycles threshold setting 0 1 Stratagene Mx3000P Mx3005P baseline setting adaptive threshold setting 0 1 Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for data analysis see instructions in Data Analysis 24 qBiomarker Somatic Mutation PCR System D qBiomarker Somatic Mutation Data Analysis 1 Excel based PCR Array Data Analysis Template Download our Excel based PCR Array Data Analysis template from the SABiosciences web site at the
27. ml tube or a multi channel reservoir Plate Format 96 well 384 well A C D F E amp G qBiomarker Probe Mastermix 1275 ul 550 ul Unamplified Genomic DNA 500 ng to 3 ug in X ul 200 ng to 1 2 ug in X ul ho o 25X 550 X ul Total volume 2550 ul 1100 ul NOTE This recipe provides an excess volume of ONLY 140 ul Very carefully add the cocktail to the qBiomarker Somatic Mutation PCR Array precisely as described below to ensure that each well receives the required volume b Loading the qBiomarker Somatic Mutation PCR Arrays i 96 Well PCR Array Formats A C D or F a CAREFULLY remove the qBiomarker Somatic Mutation PCR Array from its sealed bag b Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading c Add 25 ul of the Experimental Cocktail to each well of the PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips NOTE Change pipette tips following each addition to avoid any cross contamination between the wells or reactions d Skip the next section and proceed to Performing Real Time PCR Detection below ii 384 Well PCR Array Format E or G NOTE Each 384 well plate characterizes four samples in separate sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multi channel pipettors will allow you to properly skip rows or col
28. ng 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 10 bleach 13 A DNA Preparation and Quality Control High quality DNA is ESSENTIAL for obtaining good real time PCR results The most important prerequisite for any somatic mutation analysis experiment is consistent high quality DNA from every experimental sample Therefore the sample handling and DNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the DNA or decrease the efficiency of if not block completely the enzyme activities necessary for optimal whole genome amplification and real time PCR performance 1 Recommended Genomic DNA Preparation Method The QIAGEN QlAamp DNA Mini Kit 51304 and QlAamp DNA FFPE Tissue Kit 56404 are highly recommended for the preparation of genomic DNA samples from fresh tissues and FFPE tissue samples Ensure that samples have been treated for the removal of RNA as RNA contamination will cause inaccuracies in DNA concentration measurements DO NOT omit the recommended RNase treatment step to remove RNA If genomic DNA samples need to be harvested from biological samples where kits are not available please contact Technical Support representatives for suggestions For best results from the qBiomarker Somatic Mutation PCR Array all DNA samples
29. r accurate and reproducible PCR Array results it is very important to avoid contamination of the assay with foreign DNA especially the PCR products from previously run plates The most common sources of DNA contamination are the products of previous experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment free of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and labware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipettor barrels tube racks etc before each new use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 10 bleach to chemically inactivate and degrade any DNA 3 Do not remove the PCR Array plate from its protective sealed bag until immediately before use Do not leave labware tubes and tip boxes exposed to air for long periods of time 4 Do not open any previously run and stored PCR Array plate Removing the thin wall 8 cap strips or the adhesive film from PCR Arrays releases PCR product DNA into the air where it will contaminate and confound the results of future real time PCR experiments 5 In the event that PCR products need to be analyzed by an independent method close all tubes containi
30. s The qBiomarker Somatic Mutation PCR Arrays are available in 6 different plate formats each tailored to a specific subset of real time PCR instruments amp associated blocks Formats A C D and F are 96 well plates and Formats E amp G are 384 well plates Plate Master Mix For Real Time Instruments Format Reference Dye 96 well Bio Rad iCycler iQ 5 MyiQ MyiQ2 ABI 7500 FAST 96 well Block 7900HT FAST 96 Well 96 well ABI Standard 96 well Blocks 5700 7000 7300 7500 96 well ROX 900HT ViiA 7 Bio Rad Chromo4 MJ Research Stratagene Mx3005P Mx3000P Eppendorf ep Rox BRI StepOnePlus ViiA 7 FAST 96 well Block mox Rene CFX96 Opticon and Opticon 2 MJ Research Stratagene Mx4000 ROX ROX ROX ROX ABI 7900HT 384 well Block ViiA 7 384 well Block Bio 384 well ROX OX Rad CFX384 Roche LightCycler 480 96 well Block 96 well G ROX Roche LightCycler 480 384 well Block 384 well NOTE The format of the qBiomarker Somatic Mutation PCR Array is indicated by the last letter of the catalog number Be sure that you have the correct PCR Array format for your instrument before starting the experiment The 96 well qBiomarker Somatic Mutation PCR Arrays Formats A C D and F are shipped in sets of two 2 twenty five 25 or one hundred 100 while the 384 well qBiomarker Somatic Mutation PCR Arrays Formats E and G are shipped in sets of twenty five 25 or one hundred
31. t SABiosciences Technical Support support SABiosciences com for suggestions 15 B qBiomarker Somatic Mutation PCR Array Protocol 1 Optional Whole Genome Amplification for Genomic DNA Purified from Fresh Tissue NOTE The whole genome amplification WGA process can dramatically reduce the required amount of starting material o WGA is intended for those working with Fresh or Frozen Cell amp Tissue samples who can only isolate 5 10 ng of genomic DNA o If 200 500 ng of genomic DNA is extracted from fresh tissue and is of high quality see Section A DNA Preparation and Quality Control it is not necessary to perform this whole genome amplification step o For DNA extracted from FFPE sections we recommend skipping the WGA process NOTE The following protocol serves as a quick setup guide for the whole genome amplification process For detailed principle and instructions please refer to the handbook of the REPLI g UltraFast Mini Kit a Prepare sufficient Buffer D1 and Buffer N1 for the total number of whole genome amplification reactions see Table 1 and 2 for mixing volumes for up to 40 reactions Do not forget to include a wild type control sample Table 1 Preparation of Buffer D1 Component Volume Reconstituted Buffer DLB Nuclease free water Total volume Table 2 Preparation of Buffer N1 Stop solution Nuclease free water Total volume b Dilute genomic DNA gDNA to 10 ng ul in nuclease free wa
32. tency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure no bubbles are introduced into the wells of the PCR array NOTE Thaw genomic DNA sample and PCR Master Mix at room temperature 15 25 C Make sure they are mixed well after thawing a Experimental Cocktail Preparation According to i Table 4 if genomic DNA is amplified using REPLI g UltraFast Kit ii Table 5 if genomic DNA is not amplified However more DNA will be required to achieve the best array results For DNA from fresh tissue samples it is recommended to start with 500 ng DNA for each 96 well array or 200 ng DNA for each sample for the 384 well format qBiomarker Somatic Mutation PCR Array respectively Due to the degraded nature of DNA extracted from FFPE samples it is recommended that for FFPE DNA samples at least 500 ng up to 3 ug DNA is loaded for the 96 well format array and at least 200 ng up to 1 2 ug DNA is loaded for the 384 well format array Table 4 Preparation of PCR Array Cocktail for Amplified DNA Mix the following components in a 5 ml tube or a multi channel reservoir Plate Format 96 well 384 well A C D F E amp G qBiomarker Probe Mastermix 1275 ul 550 ul Amplified Genomic DNA 1260 yl Total volume 2550 ul 1100 ul 18 qBiomarker Somatic Mutation PCR System Table 5 Preparation of PCR Array Cocktail for Non Amplified DNA Mix the following components in a 5
33. ter c Add 1 ul gDNA into a microcentrifuge tube 16 qBiomarker Somatic Mutation PCR System d e f Add 1 ul Buffer D1 to the gDNA Mix by gentle pipetting Incubate the samples at room temperature for 3 minutes Add 2 ul Buffer N1 to the samples Mix by gentle pipetting Thaw REPLI g UltraFast DNA Polymerase on ice Thaw REPLI g UltraFast Reaction Buffer at room temperature 15 25 C vortex then centrifuge briefly Prepare a master mix on ice according to Table 3 Mix and centrifuge briefly Table 3 Preparation of Master Mix Volume per reaction REPLI g UltraFast Reaction Buffer 15 ul REPLI g UltraFast DNA Polymerase Total volume 16 ul Add 16 yl of the master mix to 4 ul of samples Incubate the samples at 30 C for 1 5 hours Incubate the samples at 65 C for 3 minutes to inactivate DNA polymerase Store amplified DNA at 20 C until used There is no need to re purify the DNA 17 2 Performing Real Time PCR NOTE Be sure to use the correct master mix for your instrument before continuing with this protocol NOTE An incorrectly chosen qBiomarker Somatic Mutation PCR Array plate format will not properly fit into your real time PCR instrument and its use will damage the instrument Be sure that you have the correct qBiomarker Somatic Mutation PCR Array format for your instrument before continuing with this protocol NOTE The accuracy and precision of your pipetting determines the consis
34. umns when adding each sample Be sure to load each sample into the correct set of wells using Figure 4 below as a guide e CAREFULLY remove the qBiomarker Somatic Mutation PCR Array from its sealed bag 19 f Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading i Load sample cocktails to appropriate wells of the qBiomarker Somatic Mutation PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips using the provided 384EZLoad Covers Catalog PA 384 338125 and the figure below as a guide a Place Cover 1 white on the plate Add 10 uL of Sample 1 cocktail to the open wells Odd number wells of rows A C E G I K M amp O Remove amp discard the cover b Place Cover 2 yellow on the plate Add 10 uL of Sample 2 cocktail to the open wells Even number wells of rows A C E G I K M amp O Remove amp discard the cover c Place Cover 3 black on the plate Add 10 uL of Sample 3 cocktail to the open wells Odd number wells of rows B D F H J L N amp P Remove amp discard the cover d Place Cover 4 red on the plate Add 10 uL of Sample 4 cocktail to the open wells Even number wells of rows B D F H J L N amp P Remove amp discard the cover Sample 1 Sample 2 1 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
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