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1. a test sample the AC for the gene of interest is Cae E Gite The AAC value is AAGE _ AC 60r The predicted copy number CN in a test sample assuming the calibrator genome has 2 copies of the is CN s 056996 891 The median genome method uses the median AC SO from a large set of samples gt 20 or a sample set without a calibrator sample The calculation method is the same as above except that the median AC from a set of samples AC 99 is used as the calibrator AC of the GOI The rationale is that the median C 9 of a set of samples will represent a normal diploid genome with 2 copies of the gene based on the assumption that the frequency for copy gain or loss for a particular locus is lower than 5096 in the sample set 50 qBiomarker Copy Number PCR Handbook 08 2012 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 ee a qBiomarker Copy Number PCR Handbook 08 2012 51 Ordering Information Product qBiomarker Copy Number PCR Arra
2. values for each sample tested The highest C value is not included in calculating the average since some samples may contain a homozygous deletion for one of the 7 genes included on the DNA QC plate This deleted gene will give a high C value The typical average C value for high quality DNA from fresh tissue samples should be 29 based on the baseline and threshold setup outlined above Samples of lower quality average value gt 29 may not give optimal results For DNA extracted from FFPE samples an average C value 32 for the lowest 6 values when using 5 ng genomic DNA 25 ul reaction volume 2 ng genomic DNA input in 10 ul reaction volume or 4 ng genomic DNA in a 20 ul reaction volume indicates sufficient quality for CNA profiling analysis Samples of lower quality average value gt 32 may not yield optimal results or may qBiomarker Copy Number PCR Handbook 08 2012 47 require more input DNA estimate the amount fold increase needed to make the average value lt 32 Note When a calibrator sample is used in copy number analysis we recommend that the average value of the calibrator sample determined as described above should be within 4 C values of that of a test sample eee 48 qBiomarker Copy Number PCR Handbook 08 2012 Appendix Data Analysis Using the AAC Method At the qBiomarker Copy Number PCR Array and Assay Data Analysis Web portal http pcrdataanalysis sabiosciences co
3. Bio Rad CFX384 F 96 well Roche LightCycler 480 96 well block G 384 well Roche LightCycler 480 384 well block R 100 well Rotor Gene Q Rotor Disc 14 qBiomarker Copy Number PCR Handbook 08 2012 Table 2 Real time PCR cyclers suitable for use with Number PCR Assays Type of instrument Real time PCR cyclers Instruments that use ROX All Applied Biosystems TaKaRa and as a passive reference dye Stratagene real time PCR cyclers Instruments that use fluorescein as a passive Bio Rad iCycler iQ5 MyiQ MyiQ2 reference dye Bio Rad MJ Research DNA Engine Opticon DNA Engine Opticon 2 and Chromo4 Instruments that do not Roche LightCycler 480 Eppendorf require a reference dye Mastercycler ep realplex 2 25 4 45 QIAGEN other instruments that do not require a reference dye qBiomarker Copy Number PCR Handbook 08 2012 15 Important Notes General precautions We recommend the following guidelines Use sterile pipet tips with filters Store and extract positive materials specimens positive controls and amplicons separately from all other reagents and add them to the reaction mix in a spatially separated facility Physically separate the workspaces used for PCR setup and post PCR processing operations Decontaminate your PCR workspace and labware pipets tube racks etc with UV light before each new use to render any contami
4. immediately may be stored wrapped in aluminum foil at 20 C for up to one week until ready to run 9 Program the real time cycler according to Table 6 7 or 8 depending on the real time cycler used If prompted by your cycler software select Absolute Quantitation to begin Note For additional help with instrument setup see the instrument specific setup instructions and protocol files at www SABiosciences com pcrarrayprotocolfiles php Table 6 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 15 95 C 1 min 60 C Perform fluorescence data collection Recommended for the following cyclers Applied Biosystems models 5700 7000 7300 7500 7700 7900HT StepOnePlus 7 Bio Rad models iCycler iQ5 MyiQ MyiQ2 CFX96 CFX384 Stratagene models Mx3000P Mx3005P Mx4000P Eppendorf Mastercycler ep realplex models 2 2S 4 45 t For Bio Rad models CFX96 and CFX384 adjust the ramp rate to 1 C s For Eppendorf Mastercyler ep realplex models 2 25 4 and 4 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 ES lt 26 qBiomarker Copy Number PCR Handbook 08
5. 05 05 17 17 05 17 17 05 05 17 17 1213141516 07 07 19 19 07 07 07 07 19 19 07 07 19 19 07 07 19 19 07 07 19 19 8 8 8 8858 8888 8 888 9 8588 88888 17 18 19 20 21 22 23 24 09 09 10 10 1 1 12 12 09 09 10 10 n 21 21 22 22 23 23 21 21 22 22 23 23 099 09 10 10 m 12 09 09 10 21 21 22 22 23 23 21 21 22 2 23 23 12 12 09 09 10 10 11 11 12 12 09 09 10 10 1 n 12 12 21 21 22 22 23 23 21 21 22 22 23 23 09 09 10 10 11 1 12 09 09 10 10 21 21 22 22 93 23 12 26 O0 00 21 21 22 22 23 23 Copy no assay MRef Figure 2 Plate layout for a 23 panel 384 well plate The numbers in the cells each represent a single well for gene 1 gene 2 etc The first set of 4 rows is repeated 3 more times so that one 384 well PCR array plate can be used to assay 4 samples qBiomarker Copy Number PCR Handbook 08 2012 19 gt 01 01 13 13 25 37 37 49 49 61 61 73 73 85 01 01 13 13 37 37 49 49 61 61 73 73 85 85 38 50 50 62 62 74 74 03 03 15 27 27 39 51 51 75 75 87 87 52 52 76 76 89 78 78 883162566 Es 12 13 14 15 16 78 78 07 07 19 19 31 31 43 43 55 55 67 67 91 91 07 07 19 19 31 31 43 43 55 55 67 67 79 79 91 91 8 888868666 ERE 888
6. 2012 Table 7 Cycling conditions for Roche LightCycler 480 Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 45 15 2918 1 60 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 Table 8 Cycling conditions for Bio Rad and Takara cyclers and other cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15 95 C 30 40 s 55C 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 305 72 T Recommended for the following cyclers Bio Rad MJ Research models Chromo4 DNA Engine Opticon DNA Engine Opticon 2 Takara TP 800 all other cyclers excluding Rotor Gene cyclers 10 Place one PCR array plate in the real time PCR cycler Use a compression pad with the optical film sealed plate formats plate formats C E F and G if recommended by the manufacturer Start the run qBiomarker Copy Number PCR Handbook 08 2012 11 12 13 1
7. 5 5 5 0 4 0 3 0 2 9 8 7 3 6 5 5 0 3 5 2 8 10 9 0 8 0 6 0 4 0 3 0 12 10 8 9 5 7 0 4 5 3 3 14 12 5 11 0 8 0 5 0 9 9 16 14 3 12 5 9 0 5 5 3 8 18 16 0 14 0 10 0 6 0 4 0 20 17 8 15 5 11 0 6 5 4 3 22 19 5 17 0 12 0 7 0 4 5 24 21 3 18 5 13 0 7 5 4 8 26 23 0 20 0 14 0 8 0 5 0 28 24 8 21 5 15 0 8 5 5 3 30 26 5 23 0 16 0 9 0 5 5 32 28 3 24 5 17 0 9 5 5 8 42 qBiomarker Copy Number PCR Handbook 08 2012 Appendix A Quality Control of Genomic DNA Using the DNA QC Plate Sample DNA quality can affect the performance of qBiomarker Copy Number PCR Arrays and Assays For DNA extracted from FFPE samples different degrees of cross linking and fragmentation may result in suboptimal PCR results When uncertain of the sample quality it is recommended to check the DNA quality first using the DNA QC Plate The DNA QC Plate is designed to measure the C values of 7 reference genes When the DNA is highly cross linked or fragmented the values from these 7 genes will be much higher than those from the same amount of high quality DNA DNA QC Plates are available in formats A C D and F 96 well plates Figure 6 formats E G 384 well plates see Product Sheet or Web site for plate layout or format R Rotor Disc 100 see Product Sheet or Web site for plate layout Each 96 well format DNA QC Plate can be used for quality control of 12 DNA samples Each 384 well format DNA QC Plate can be used for quality control of 48 DNA samples Each Rotor Di
8. Do not use DEPC treated water Use high quality nuclease free water If precipitates are present in the mastermix tube warm the reagents at 42 for 1 min and vortex briefly to dissolve Repeat if necessary Procedure 1 Briefly centrifuge the qBiomarker SYBR Mastermix 10 15 s to bring the contents to the bottom of the tube Note As the qBiomarker SYBR 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 a reaction mix according to Table 9 for each sample For genomic DNA from fresh tissue samples it is recommended to start with 0 4 1 0 ug DNA for each sample Due to the fragmented nature of DNA extracted from FFPE samples it is recommended to use 0 8 1 2 ug DNA for each sample Table 9 Preparation of reaction mix Array format Rotor Disc 100 SYBR Mastermix 1150 ul 0 4 1 0 ug fresh 0 8 1 2 ug FFPE Nuclease free water Variable Total volume 2300 pl This volume provides an excess of 300 ul per sample to allow for some imprecision in pipetting 3 Carefully remove the PCR array Rotor Disc from its sealed bag Slide the Rotor Disc into the Rotor Disc 100 Loading Block using the tab at position A1 and the tube guide holes 4 Add 20 pl reaction mix to each well of the Rotor Disc Proceed to step 5 qBiomarker Copy Number PCR Handbook 08 2012 31 Note Change pipet t
9. M and Remove and discard the cover 384bEZLoad Cover 3 black Place 384EZload Cover black on the plate Add 10 ul sample 3 reaction mix to the open wells odd numbered wells of rows B D F H J L N and P Remove and discard the cover 384EZLoad Cover 4 red Place 384EZload Cover 4 red on the plate Add 10 ul sample 4 reaction mix to the open wells even numbered wells of rows B D F H J L N and P Remove and discard the cover 384 well 95 gene panel Formats E Add 10 ul reaction mix to each well of the PCR array plate using a multichannel pipet with 8 tips Each 384 well plate 95 gene panel characterizes one sample in 4 sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multichannel pipets should allow you to properly load the sample into every other row and column 6 Carefully but tightly seal the PCR array plate with the Optical Thin Wall 8 Cap Strips plate formats A and D or with the Optical Adhesive Film plate formats C E F and G qBiomarker Copy Number PCR Handbook 08 2012 25 7 Remove any air bubbles the wells of the PCR array plate by centrifuging the plate at 1000 rpm for 1 min 96 well plate formats A C D F or 2000 rpm for 2 min 384 well plate formats E G 8 Place the PCR array plate on ice while setting up the PCR cycling program in step 9 Note PCR array plates containing reaction mix that will not be processed
10. based software Note qBiomarker Copy Number PCR Array Data Analysis Software is available at http pcrdataanalysis sabiosciences com cnv 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 Note If your cycler does not have a default melting curve 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 Plates 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 plate to room temperature 15 252 place it in the real time cycler and run the melting curve analysis program qBiomarker Copy Number PCR Handbook 08 2012 Note Visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed qBiomarker Copy Number PCR Array Removing the Optical Thin Wall 8 Cap Strips or the Optical Adhesive Film from a qBio
11. buffers treated with diethylpyrocarbonate DEPC DNA quality control All DNA samples should show consistent quality either by UV spectrophotometric measurements or by performance in real time PCR using the DNA QC Plate Measuring DNA concentration and purity by UV spectrophotometry Dilute samples and measure absorbance 10 mM Tris Cl pH 8 0 An absorbance reading of 1 0 at 260 nm 1 cm detection path corresponds to DNA concentration of 50 ug ml All DNA samples should meet the following criteria Concentration as measured by Aseo should be greater than 10 ug ml ratio should be greater than 1 8 ratio should be greater than 1 7 Measuring DNA quality with the DNA QC Plate DNA quality and consistency can be checked more reliably with the DNA QC Plate by real time PCR measuring 7 reference genes For a detailed procedure see Appendix A Quality Control of Genomic DNA Using the DNA QC Plate page 43 qBiomarker Copy Number PCR Array plate layouts Each qBiomarker Copy Number PCR Array shipment includes the array plates discs and either 12 Optical Thin Wall 8 Cap Strips plate formats A and D one Optical Adhesive Film plate formats C E F and G or one Rotor Disc Heat Sealing Film disc format R per plate disc Each 384 well array formats E and G also includes one set of four 384EZLoad Covers cat no 338125 for each plate Each 384EZLoad Cover is for single us
12. e g QIAGEN cat nos 129114 129115 or 129117 do not use water treated with diethylpyrocarbonate DEPC Real time PCR cycler see Tables 1 and 2 pages 14 and 15 Single and multichannel pipets with nuclease free tips Nuclease free microcentrifuge tubes Centrifuge and rotor for PCR array plates Crushed ice Optional DNA QC Plate QIAGEN please inquire for cat no for assessing DNA quality Optional RT PCR Array Loading Reservoir cat no 338162 to assist in loading PCR plates Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations qBiomarker Copy Number PCR Handbook 08 2012 13 Table 1 Real time PCR cyclers suitable for use with Number PCR Arrays Plate format Plate type Real time PCR cyclers Applied Biosystems models 5700 7000 7300 7500 7900HT 96 well block ViiA 7 96 well block Bio Rad MJ Research Chromo4 iCycler iQ5 MyiQ MyiQ2 Stratagene models Mx3000P Mx3005P Eppendorf Mastercycler ep realplex models 2 25 4 4S TaKaRa TP 800 Applied Biosystems models 7500 Fast 96 well 96 well 7900HT Fast 96 well block StepOnePlus ViiA 7 Fast 96 well block Bio Rad 96 Bio Rad MJ Research models A 96 well D 96 well DNA Engine Opticon DNA Engine Opticon 2 Stratagene Mx4000 384 well Applied Biosystems models 7900 384 well block 7 384 well block
13. gt gt gt gt gt gt 1 gt gt 77 gt w gt gt gt gt gt gt gt gt gt gt gt gt e gt gt gt gt gt gt gt gt gt gt gt e gt gt Cover 2 yellow for sample 2 2 D 2 22 2 D po Do 2 2 2 2 2 2 2 2 2 2 0 0 0 3143 43434343434 3434343434 343 4 34 3 A 3A 3 A3 A 3 A 3A a A 3A 2 gt gt gt gt gt gt gt gt 2 2 2 2 2 2 2 343434343434343434343434 34343434343434343434343 4 1B gt 341394343 4343943 43434943434 1B 2 343 434343434343434343434 343434343434343434343434 Cover 3 black for sample 3 1 15 1 15 15 1 5 112112112 1121112112112 1
14. targets see Appendix B Data Analysis Using the AAC Method page 49 Each qBiomarker Copy Number PCR Array contains a panel of qBiomarker Copy Number PCR Assays for a set of selected GOls or ROIs Four replicates of each assay are included to increase the accuracy of copy number calls using statistical analysis The arrays are available in 96 well plate 384 well plate and Rotor Disc formats A 96 well plate array and a Rotor Disc array each contain 4 replicates of 24 genes 23 target genes plus MRef for one sample and a 384 well plate array can either contain 16 replicates of 24 genes 23 target genes plus MRef for 4 samples or 4 replicates of 96 genes 95 target genes plus MRef for one sample For more details see page 17 Description of protocols The qBiomarker Copy Number PCR procedure see workflow page 12 starts with genomic DNA purification After quality control the DNA is mixed with the ready to use qBiomarker SYBR Mastermix The mixture is aliquoted into each well of the array plate containing preloaded gene specific primers After real time PCR the copy number profile of a particular sample is determined using the AAC method measuring the difference in values target gene reference assay between a test sample genome and a reference genome see Appendix Data Analysis Using the AAC Method page 49 The simplicity of the qBiomarker Copy Number PCR Array format and protocol allows routine CNA
15. 121121121121 2 UEJEREJEREJEREJER ESEEEJIEREJERES UEEJEEEJEGEJERE imn nm mnm m mm mBmm mBm mBm nDmu 3 4 15 15 15 15 11212 112 1121121112112 1121121112 112 112112 5 15 15 15 pc J H H H H HH EH H H EB EH ZV Zi VIS US VZV 15 amp 8 11211121112 1121112111211 2 112 11211 21112 1 12 1121112111211 12111211 121112111211 1211121112112 1 2 1 2 1 2 1 2 1 2 1 2 1 2 112 1 2 1 2 1 2 1 2 1 1 5 0 0 8 8 H H H H H H H H H BH EB B Cover 4 red for sample 4 Figure 5 Loading 384 well 23 gene panel formats E G 384EZLoad Cover 1 white Place 384EZload Cover 1 white on the plate Add 10 ul sample 1 reaction mix to the open wells odd numbered wells of rows A C M and Remove and discard the cover 384EZLoad Cover 2 yellow Place 384EZload Cover 2 yellow on the plate Add 10 sample 2 reaction mix to the open wells even numbered wells of rows A C E I
16. 4 15 28 After the run is finished calculate the threshold cycle for each well using the real time cycler software as described in the following steps Note If using the Roche LightCycler 480 there are 2 options for data analysis using the second derivate max setting in this case there is no need to calculate the or using Fit Points in this case the should be defined manually as described in step 13 Define the baseline by choosing the automated baseline option if the cycler has the adaptive baseline function If the cycler does not have the adaptive baseline function set the baseline manually To set the baseline manually use the linear view of the amplification plots to determine the earliest visible amplification Set the cycler to use the readings from cycle number 2 up to 2 cycles before the earliest visible amplification Manually define the threshold by using the log view of the amplification plots Choose a threshold value above the background signal but within the lower one third to lower half of the linear phase of the amplification plot Note Ensure that the threshold values are the same across all qBiomarker Copy Number PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays Export the values for all wells to a blank Excel spreadsheet for use with the qBiomarker Copy Number PCR Array Data Analysis Web
17. 8686 EE 08 17 18 192021222324 09 09 21 21 33 33 45 45 57 57 69 69 81 81 93 93 09 09 21 21 33 33 45 45 57 57 69 69 81 81 93 93 10 88 5565 8 8 94 94 1 11 23 23 35 35 47 47 59 59 71 71 83 83 95 95 11 11 23 23 35 35 47 47 59 59 71 12 N 3 3 E ERR 12 N 3 3 no assay E MRef Figure 3 Plate layout for 95 panel on 384 well plate The numbers in the cells each represent a single well for gene 1 gene 2 etc One 384 well PCR array plate can be used to assay one sample qBiomarker Copy Number PCR Handbook 08 2012 20 100 well Rotor Disc layout for 23 gene panel one disc for one sample has 4 replicates of 23 genes and a multi copy reference assay MRef arranged as shown in Figure 4 Copy no assay Bl 3 Figure 4 Disc layout for a 23 gene panel on a 100 well Rotor Disc One Rotor Disc can be used to assay one sample qBiomarker Copy Number PCR Handbook 08 2012 21 Protocol PCR Using qBiomarker Copy Number Array Formats A C D E F and G This protocol is for use with qBiomarker Copy Number PCR Arrays 96 well and 384 well plates For use of qBiomarker Copy Number PCR Arrays Rotor Discs see PCR Using qBiomarker Copy Number PCR Arrays Format page 30 For use of individual qBiomarker Copy Number PCR Assays see PCR Using qBiomark
18. August 2012 qBiomarker Copy Number Handbook qBiomarker Copy Number PCR Array qBiomarker Copy Number PCR Assay For real time PCR based copy number alteration and variation analysis 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 Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and 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 Array Contents 4 Assay Contents 5 Mastermix Contents 6 Shipping and Storage 7 Intended Use 7 Product Warranty and Satisfaction Guarantee 8 Technical Assistance 8 Safety Information 9 Quality Control 9 Introduction 10 Principle and procedure 11 Description of protocols 1 Equipment and Reagents to Be Supplied by User 13 Important Notes 16 General precautions 16 DNA purification 16 DNA quality control 17 qBiomarker Copy Number PCR Array plate layouts 17 Protocols PCR Using qBiomarker Number PCR Array Formats A C D E F and G 22 E PCR Using qBiomarker Copy Number PCR Array Format R 30 E PCR
19. Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15 95 30 40 55310 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 30 T2 Recommended for the following cyclers Bio Rad MJ Research models Chromo4 DNA Engine Opticon DNA Engine Opticon 2 Takara TP 800 all other cyclers 8 Place the PCR plate disc in the real time PCR cycler Use a compression pad with the optical film sealed plate formats plate formats C F and if recommended by the manufacturer Start the run 9 After the run is finished calculate the threshold cycle C for each well using the real time cycler software For assays performed using a Rotor Gene cycler follow steps 8 and 9 page 32 and 33 For assays performed using all other cyclers follow steps 11 13 page 28 10 Export the C values for all wells to a blank Excel spreadsheet for use with the qBiomarker Copy Number PCR Assay Data Analysis Web based software Note qBiomarker Copy Number PCR Assay Dota Analysis Software is available at http pcrdataanalysis sabiosciences com cnv 11 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 r
20. QIAGEN GlAgility DNeasy MinElute Rotor Disc Rotor Gene QIAGEN Group Applied Biosystems ROX StepOnePlus Applera Corporation or its subsidiaries Bio Rad Chromo4 CFX96 CFX384 DNA Engine Opticon iCycler IQ MyiQ Bio Rad Laboratories Inc Eppendorf Mastercycler Eppendorf AG Excel Microsoft Corporation SYBR Molecular Probes Inc LightCycler Roche Roche Group Mx3005P Mx3000P 4000 Stratagene Agilent Technologies Limited License Agreement for qBiomarker Copy Number PCR Arrays qBiomarker Copy Number PCR Assays and qBiomarker SYBR Mastermixes Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this 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 protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the ri
21. Using qBiomarker Copy Number PCR Assays 34 Troubleshooting Guide 40 Appendix A Quality Control of Genomic DNA Using the DNA QC Plate 43 Appendix B Data Analysis Using the AAC Method 49 References 51 Ordering Information 52 qBiomarker Copy Number PCR Handbook 08 2012 3 Array Contents qBiomarker Copy Number PCR Arrays Catalog no 337802 Number of samples 2 12 24 4 16 64 96 well plates A C D F formats 2 12 24 384 well plates formats 1 4 16 Rotor Discs R format 2 12 24 Optical Thin Wall 8 Cap Strips 12 per plate A D formats only 24 144 288 Optical Adhesive Film 1 per plate C E F G formats only N 12 24 1 4 16 Rotor Disc Heat Sealing Film 1 per Rotor Disc R format only 2 12 24 384EZLoad Covers 1 set of 4 per 16 plate formats only i i 7 ML E Handbook 1 1 1 1 1 1 4 qBiomarker Copy Number PCR Handbook 08 2012 Custom qBiomarker Number PCR Arrays Catalog no 337801 Number of samples Varies Varies Varies Varies 96 well plates A C D F formats 12 24 384 well plates E G formats 6 24 Rotor Discs format 12 24 Optical Thin Wall 8 Cap Strips 12 per plate A D formats only 152 255 E Optical Adhesive Film 1 per plate C E F G formats only ne 6 ag Rotor Disc Heat Sealing Film 1 per 12 24 E Rotor Disc R format only Handbook 1 1 1 1 Assay Contents qBiom
22. able at http pcrdataanalysis sabiosciences com cnv 11 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 Note Melting curve analysis can be added during creation of the Rotor Gene Q PCR program Note For Rotor Gene Q melting curve analysis settings refer to the Instrument Setup Guide at www SABiosciences com pcrarrayprotocolfiles php Note Rotor Discs 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 plate to room temperature 15 252 place it in the real time cycler and run the melting curve analysis program Note Visually inspect the Rotor Disc after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed qBiomarker Copy Number PCR Array Removing the film from qBiomarker Copy Number PCR Arrays releases PCR product into the air where it may contaminate and affect the results of future real time PCR experiments qBiomarker Copy Number PCR Handbook 08 2012 33 Protocol PCR Using qBiomarker Copy Number Assays This protocol is for use with qBiomarke
23. ach well of the PCR array plate as follows Note Perform pipetting steps as precisely as possible to ensure that each well receives the required volume Note Change pipet tips following each addition to avoid any cross contamination between the wells 96 well 23 gene panel Formats A C D F Add 25 ul reaction mix to each well of the PCR array plate using a multichannel pipet with 8 tips 384 well 23 gene panel Formats E Add 10 ul reaction mix to each well of the PCR array plate using a multichannel pipet with 8 tips Each 384 well plate 23 gene panel characterizes 4 samples in separate sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multichannel pipets should allow correct loading of samples into every other row and column Be sure to load each sample into the correct set of wells using the 384EZLoad Covers provided and see Figure 5 as a guide 24 qBiomarker Copy Number PCR Handbook 08 2012 Cover 1 white for sample 1 2 2 2 2 gt gt gt gt gt gt gt e gt 77 gt gt gt gt gt 77 gt 7 gt gt gt 77 gt e gt gt gt gt gt gt 77 gt gt gt gt 1 gt gt
24. ackground signal but within the lower half to one third of the linear phase of the amplification plot 46 qBiomarker Copy Number PCR Handbook 08 2012 A12 For format To define the baseline value select Ignore First Manually define the threshold value by using the log view of the amplification plots Select a threshold value above the background signal The threshold value should be in the lower half to one third of the linear phase of the amplification plot A threshold setting of 0 03 is recommended as a reference Fluorescent signal from the initial cycles may not be representative of the remainder of the run Therefore better results may be achieved if the initial cycles are ignored Up to 5 cycles can be ignored Table 19 shows typical settings for selected real time PCR cyclers Actual settings may differ Table 19 Typical baseline and threshold settings Instruments Baseline Threshold Applied Biosystems 7900HT 5 18 cycles 0 1 Applied Biosystems 7500 5 18 cycles 0 1 Stratagene Mx3000P Mx3005P Varies 0 1 A13 Export the resulting C values for all wells to a blank Excel spreadsheet Data analysis To determine the quality of DNA samples based on the results first ensure that the of the SMPC assay for each sample is consistent at 22 formats A C D E F G or 21 format R If not adjust the baseline and threshold setting to achieve that value Then calculate the average of the lowest 6
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26. and CNV profiling in any research laboratory with access to a real time PCR cycler e qBiomarker Copy Number Handbook 08 2012 11 Purify DNA using the QlAamp DNA Mini Kit DNeasy Blood amp Tissue Kit or DNA FFPE Tissue Kit approximately 30 minutes Y Measure DNA concentration and purity by UV spectrophotometry Y Optional Run quality control check using DNA QC Plate Y Set up and run real time PCR using qBiomarker Copy Number PCR Arrays or Assays 2 hours M Analyze data using the online analysis template 10 20 minutes 12 qBiomarker Copy Number PCR Handbook 08 2012 Equipment and Reagents to Be Supplied 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 In addition to the qBiomarker Copy Number PCR Array or Assay and qBiomarker SYBR Mastermix the following are required For gBiomarker Copy Number PCR Assays 96 well or 384 well PCR plate with Optical Thin Wall 8 Cap Strips or Optical Adhesive Film or Rotor Disc with Rotor Disc Heat Sealing Film or PCR tubes Purified DNA samples see DNA purification page 16 Spectrophotometer cuvettes and nuclease free Tris Cl pH 8 0 for measuring DNA quality and quantity Nuclease free water
27. arker Copy Number PCR Assays Catalog no 337812 Number of samples 200 qBiomarker Copy Number PCR Assay provided in solution 200 ul Handbook 1 qBiomarker Copy Number Handbook 08 2012 5 Mastermix Contents qBiomarker SYBR 2 12 24 8 25 ml Fluor Mastermix Catalog no 337830 337832 337833 337831 337839 Number of array 2x 12x 24 x 4x 2000 x reactions 96 well 96 well 96 well 384 25 pl well reactions 2x Mastermix 2x 12x 24 x 8 x 25 ml containing HotStart DNA 1 35 1 35 ml 1 35 ml 1 35 ml Taq Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP SYBR Green dye Fluorescein passive reference dye Suitable for use with the following real time cyclers Bio Rad models iCycler iQ 5 MyiQ MyiQ2 qBiomarker SYBR 2 12 24 8 25 ml ROX Mastermix Catalog no 337820 337822 337823 337821 337829 Number of array 2x 12x 24 x 4x 2000 x reactions 96 well 96 well 96 well 384 25 pl well reactions 2x Mastermix 2x 12x 24 x 8x 25 ml containing HotStart 1 35 1 35 ml 1 35 ml 1 35 ml Taq Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP SYBR Green dye ROX passive reference dye 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 bl
28. channel pipet Note Change pipet tips following each addition to avoid any cross contamination between the wells Tightly seal the PCR plate with Optical Thin Wall 8 Cap Strips or Optical Adhesive Film If using a Rotor Disc or Strip Tubes tightly seal using the Rotor Disc Heat Sealing Film or caps Remove any air bubbles in any of the plate wells by centrifuging the plate at 1000 rpm for 1 min 96 well plate formats A C D F 2000 rpm for 2 min 384 well plate formats E This step is not necessary for Rotor Disc format R Place the PCR plate on ice while setting up the PCR cycling program in step 7 Program the real time cycler according to Table 12 13 14 or 15 depending on the real time cycler used If prompted by your cycler software select Absolute Quantitation to begin Note For additional help with instrument setup see the instrument specific setup instructions and protocol files at www SABiosciences com pcrarrayprotocolfiles php qBiomarker Copy Number PCR Handbook 08 2012 Table 12 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 15 95 1 60 Perform fluorescence data collection Recommended for the following cyclers Applied Biosystems models 5700 7000 7300 7500 7700 7900HT StepOnePlus 7 Bio Rad m
29. cler according to the manufacturer s instructions using the conditions outlined in Table 18 Table 18 Cycling conditions Cycles Duration Temperature Comments 10 min 95 Initial PCR activation step AO 15s 95 Denaturation step 60 60 Annealing and extension Detect and record FAM fluorescence from every well during the annealing extension step of each cycle A9 Place the DNA QC Plate in the real time PCR cycler Use a compression pad with the optical film sealed plate formats plate formats C E F and G if recommended by the manufacturer For format R insert the Rotor Disc 100 into the Rotor Disc 100 Rotor and secure with the Rotor Disc 100 Locking Ring Start the run A10 After the run is finished calculate the threshold cycle for each well using the real time cycler software as described in the following steps Note Ensure the settings are the same across all PCR assay runs in the same analysis A11 For formats A C D E F G We highly recommend manually setting the baseline and threshold values see Table 19 for examples To define the baseline value use the linear view of the amplification plots and set the instrument to use the readings from cycle 5 up to 2 cycles before the earliest visible amplification usually around cycle 15 but not greater than cycle 20 To define the threshold value use the log view of the amplification plots and place the threshold value above the b
30. d more information 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 8 qBiomarker Copy Number PCR Handbook 08 2012 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com Support safety where you can find view and print the SDS 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 qBiomarker Copy Number PCR Arrays qBiomarker Copy Number PCR Assays and qBiomarker SYBR Mastermixes is tested against predetermined specifications to ensure consistent product quality qBiomarker Copy Number PCR Handbook 08 2012 9 Introduction qBiomarker Copy Number PCR Arrays and Assays are designed for highly sensitive detection and profiling of copy number alterations CNAs and copy number variations CVs CNAs are an important type of genomic aberration in cancer cells Gene amplifications which activate oncogenes and de
31. e only qBiomarker Copy Number PCR Handbook 08 2012 17 Plate layouts The 96 well plate layout for a 23 gene panel one plate for one sample has 4 replicates for 23 genes and a multi copy reference assay MRef arranged as shown in Figure 1 123456 7 8 9 1011 12 13 14 15 16 177 18 19 20 21 22 23 01 02 03 04 05 06 07 08 09 10 11 1 12 L 13 14 15 16 17 18 19 20 21 22 23 5 01 02 03 04 05 06 07 08 09 10 11 12 E L D 13 14 15 16 17 18 19 20 21 22 23 8 E 02 03 04 05 06 07 08 09 10 11 12 tr 01 02 03 04 05 06 07 08 09 10 11 1 12 1314 15 16 17 18 19 20 21 22 23 Figure 1 Plate layout for a 23 gene panel 96 well plate The numbers in the cells each represent a single well for gene 1 gene 2 etc The 384 well plate layout for a 23 gene panel one plate for 4 samples has 16 replicates for 23 genes and multi copy reference assay MRef arranged as shown in Figure 2 A set of 4 rows contains 4 replicates for 23 genes The 384 well plate layout for a 95 gene panel one plate for one sample has 4 replicates of 95 genes and a multi copy reference assay MRef arranged as shown in Figure 3 18 qBiomarker Copy Number PCR Handbook 08 2012 01 13 13 01 13 13 01 01 13 13 01 13 u wOZzrn mxc rommonnouvu 13 01 01 13 13 01 01 13 13 01 01 13 13 01 01 13 13 05 05 17 17
32. eal time cycler software A single peak should appear in each reaction Note If your cycler does not have a default melting curve 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 38 qBiomarker Copy Number PCR Handbook 08 2012 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 Plates discs 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 plate disc to room temperature 15 25 C place it in the real time cycler and run the melting curve analysis program Note Visually inspect the plate disc after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed qBiomarker Copy Number PCR Assay plates discs Removing the strips or film releases PCR product into the air where it may contaminate and affect the results of future real time PCR experiments _ _ qBiomarker Copy Number Handbook 08 2012 39 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more inf
33. er Copy Number PCR Assays page 34 Important points before starting 22 Before beginning the procedure read Important Notes pages 16 21 Make sure that the sample cell population contains at least 2596 tumor cells for CNA profiling analysis A calibrator sample is a DNA sample with known copy number for the targets of interest When a calibrator sample or samples are used we recommend that if possible the calibrator sample is from the same tissue type as the test sample We also recommend that the DNA quality of the calibrator sample should be comparable to that of the test sample see Appendix A Quality Control of Genomic DNA Using the DNA QC Plate page 43 for details of assessment of sample quality For fresh frozen test samples DNA from a fresh frozen sample should also be used as a calibrator sample For FFPE test samples there are 2 options for a calibrator sample DNA from a fresh frozen sample can be used as a calibrator sample although the final copy number call may be largely influenced by the quality difference between the test sample and the calibrator sample Alternatively DNA from an FFPE sample can be used as a calibrator sample In this case it is preferable to include multiple calibrator samples in the study Take time to familiarize yourself with the real time PCR cycler before starting the protocol Read the cycler user manual and follow the manufacturer s instructions for proper cycler operation and
34. ghts of third parties 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 purchaser and user of the 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 qiagen com 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 6 127 155 5 677 152 claims 1 to 23 only 5 773 258 claims 1 and 6 only and claims outside the US corresponding to expired US Patent No 5 079 352 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 and no right to perform commercia
35. he Results button at the top of the page Click the Copy Number button or p value button Sample copy number calls and statistical significance are displayed in tabular format Click Export to export the results Copy number call calculation methods The real time PCR for each gene of interest is performed at the same time as the PCR for the reference gene MRef In theory the difference between the value of a C499 and that of the multi copy reference assay C i e C 9 C M amp f AC S is the same across all samples with equivalent qBiomarker Copy Number PCR Handbook 08 2012 49 copies of the GOI even if the amount of DNA used the PCR varies between samples Therefore the AAC method can be used to calculate the copy number when a 2 value is known for a known copy number of the GOI in a reference genome e g 2 for a normal diploid genome There 2 ways to calculate the for a GOI for copy number calls in test samples based upon the 2 types of reference used The calibrator genome method compares test results with the from a calibrator genome normal diploid genome AC 99 This method can be used when a calibrator sample is available The normal diploid calibrator DNA is tested on the same type of array or assay as used for test samples The AC for the calibrator genome AC is calculated as follows AC 99 C C GOHC
36. ing the batch numbers from your assays and arrays t Larger sizes available please inquire 52 qBiomarker Copy Number PCR Handbook 08 2012 Product Contents Cat no 384EZLoad Covers 4 Pack of 4 color coded covers for 338125 loading 384 well plates DNA purification kits QlAamp DNA Mini Kit For 50 DNA preps 50 QlAamp Mini 51304 50 Spin Columns QIAGEN Proteinase K Reagents Buffers Collection Tubes 2 ml QlAamp DNA Mini Kit For 250 DNA preps 250 QlAamp Mini 51306 250 Spin Columns QIAGEN Proteinase K Reagents Buffers Collection Tubes 2 ml DNeasy Blood amp Tissue For 50 DNA preps 50 DNeasy Mini 69504 Kit 50 Spin Columns Proteinase K Buffers Collection Tubes 2 ml DNeasy Blood amp Tissue For 250 DNA preps 250 DNeasy Mini 69506 Kit 250 Spin Columns Proteinase K Buffers Collection Tubes 2 ml QlAamp DNA FFPE For 50 DNA preps 50 QlAamp 56404 Tissue Kit 50 MinElute Columns Proteinase K Buffers Collection Tubes 2 ml RNase 17 500 U 2 5 ml 100 mg ml 7000 units ml 19101 solution 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 ee e qBiomarker Copy Number PCR Handbook 08 2012 53 Notes 54 qBiomarker Copy Number PCR Handbook 08 2012 Trademarks
37. ions 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 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 qBiomarker Copy Number PCR Arrays qBiomarker Copy Number PCR Assays qBiomarker SYBR Mastermixes 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 an
38. ips following each pipetting step to avoid cross contamination between the wells Note Reaction mix can be dispensed manually or using the GlAgility www giagen com goto QIAgility Note Although wells 97 100 do not contain assays it is essential to add reaction mix for optimized balancing of the Rotor Disc Carefully seal the Rotor Disc with Rotor Disc Heat Sealing Film using the Rotor Disc Heat Sealer For detailed instructions see the Rotor Gene Q User Manual Note The Rotor Disc containing PCR components mix may be stored at 20 wrapped in aluminum foil for up to one week if desired Program the real time cycler according to Table 10 Note For additional help with instrument setup see the instrument specific setup instructions and protocol files at www SABiosciences com pcrarrayprotocolfiles php Table 10 Cycling conditions for Rotor Gene cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15 95 30 60 Perform fluorescence data collection 32 Insert the Rotor Disc into the Rotor Disc 100 Rotor and secure with the Rotor Disc 100 Locking Ring Start the run For detailed instructions see the Rotor Gene Q User Manual After the run is finished calculate the threshold cycle for each well using the real time cycler software To define the baseline select Dynamic Tube default analysis setting to ens
39. l 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 The purchase of this product includes a limited non transferable right to use the purchased amount of the product to perform Applied Biosystems patented Passive Reference Method for the purchaser s own 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 For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 2012 QIAGEN all rights reserved n www qiagen com Australia techservice au qi
40. letions which inactivate tumor suppressor genes are important CNAs that affect cancer related genes addition to other genomic changes such as point mutations translocations and inversions Most of the cancer related genes affected by CNAs have been defined as key genes in cancer signaling pathways involved in carcinogenesis and cancer progression CNVs are an important source of genetic diversity De novo locus specific mutation rates appear to be much higher for CNVs than for other forms of genetic variation such as SNPs CNVs can cause Mendelian diseases through various molecular mechanisms such as gene dosage gene disruption gene fusion and positional effect and they can also be associated with complex diseases and traits and profiling Quantitative analysis of CNAs or CNVs using qBiomarker Copy Number PCR Arrays is a useful tool for study of the molecular mechanisms of disease and genetic traits and also provides the potential for discovery of novel biomarkers qBiomarker Copy Number PCR Arrays are available to target key CNAs of cancer related genes that are known to occur in specific tissue types gene pathways and functional groups The genes are selected from peer reviewed publications based on their frequency their function in cancer signaling pathways and their association with a cancer phenotype or progression In addition qBiomarker Copy Number PCR Arrays are available for CNV profiling that focus
41. m cnv data can be entered and the Web based software will automatically perform quantification using the AAC method as described below Results are presented in a tabular format Data analysis using the qBiomarker Copy Number PCR Array and Assay online data analysis tool B1 Go to hitp perdataanalysis sabiosciences com cnv to access the online data analysis tool Select the real time PCR cycler used i e Rotor Gene cycler or Non Rotor Gene cycler B2 Upload the raw C data In the Load Excel file field click the Browse button In the pop up window that appears locate the Excel file containing the data to be analyzed and then select array assay layout target assay number and replicate reaction number and click Open Note The target assay number should include the MRef assay Note The input Excel file should follow the format of the Sample File provided B3 Click the Upload button The uploaded raw data is displayed B4 Assign the Calibrator Group if any Assign sample groups B5 Click the Update button in the upper left corner B6 Click the QC button B7 If it is necessary to remove a sample that does not pass quality control indicated on QC page as Inquire remove the sample data from the input Excel file and return to step B2 to reupload the raw data Alternatively to keep any sample that does not pass quality control proceed to step B8 B8 Click t
42. maintenance The chemically modified hot start enzyme along with other proprietary chemical components in the qBiomarker SYBR Mastermix is essential for successful function of the arrays Do not replace any of these reagents with other commercial counterparts If a calibrator DNA sample is included it should be run in the same real time PCR cycler as the test samples PCR array plates should only be used in the compatible real time PCR cyclers listed in Table 1 page 14 The PCR array plates will not fit properly into incompatible real time PCR cyclers and may cause damage to the cycler qBiomarker Copy Number PCR Handbook 08 2012 Pipetting accuracy and precision affects the consistency of results Be sure that all pipets and instruments have been checked and calibrated according to the manufacturer s recommendations Make sure that no bubbles are introduced into the wells of the PCR array during pipetting Do not use DEPC treated water Use high quality nuclease free water If precipitates are present in the mastermix tube warm the reagents at 42 for 1 min and vortex briefly to dissolve Repeat if necessary Procedure 1 Briefly centrifuge the qBiomarker SYBR Mastermix 10 15 s to bring the contents to the bottom of the tube Note As the qBiomarker SYBR Mastermix contains HotStart DNA Taq Polymerase that is active only after heat activation reactions can be prepared at room temperature 15 25 C 2 P
43. marker Copy Number PCR Array releases PCR product into the air where it may contaminate and affect the results of future real time PCR experiments qBiomarker Copy Number PCR Handbook 08 2012 29 Protocol PCR Using qBiomarker Copy Number Array Format R This protocol is for use with qBiomarker Copy Number PCR Arrays in 100 well Rotor Discs For use of qBiomarker Copy Number PCR Arrays in 96 well and 384 well plates see PCR Using qBiomarker Copy Number PCR Arrays Formats A C D E F and page 22 For use of individual qBiomarker Copy Number PCR Assays see PCR Using qBiomarker Copy Number PCR Assays page 34 Important points before starting Before beginning the procedure read Important Notes pages 16 21 Make sure that the sample cell population contains at least 25 tumor cells for CNA profiling analysis Acalibrator sample is a DNA sample with known copy number for the targets of interest When a calibrator sample or samples are used we recommend that if possible the calibrator sample is from the same tissue type as the test sample We also recommend that the DNA quality of the calibrator sample is comparable to that of the test sample see Appendix A Quality Control of Genomic DNA Using the DNA QC Plate page 43 for details of assessment of sample quality For fresh frozen test samples DNA from a fresh frozen sample should also be used as a calibrator sample For FFPE test samples there are 2
44. nated DNA ineffective in PCR through the formation of thymidine dimers or with 10 bleach to chemically inactivate and degrade any DNA Do not remove the PCR array plate from its protective sealed bag until immediately before use Thaw all components thoroughly at room temperature 15 25 before starting an assay When thawed mix the components by pipetting repeatedly up and down or by pulse vortexing and centrifuge briefly DNA purification The kits from QIAGEN shown in Table 3 are recommended for genomic DNA purification from the indicated sample types for use with qBiomarker Copy Number PCR Arrays or Assays Table 3 DNA purification kits recommended for use with qBiomarker Copy Number PCR Assays and Arrays Catalog number Sample material Nucleic acid isolation kit QIAGEN Fresh or frozen QlAamp DNA Mini Kit 51304 or 51306 tissues or cultured cells DNeasy Blood amp Tissue Kit 69504 or 69506 joy GlAamp DNA FFPE Tissue paratffin embedded Kit P 56404 FFPE tissues 16 qBiomarker Copy Number PCR Handbook 08 2012 Carry out the DNA purification according to the instructions the kit handbooks but with the following changes to the kit protocols Always perform the optional RNase A digest to remove RNA RNA contamination will cause inaccuracies in DNA concentration measurements with nuclease free water or the elution buffer provided the DNA purification kit Do not use water or
45. o allow for imprecision in pipetting Table 17 Preparation of reaction mix for 8 4 replicate PCRs Number of reactions Rotor 96 well 384 well Disc plate plate 100 qBiomarker Probe Mastermix 2x included with DNA QC Plate mu 40 16 ng 32 Nuclease free water Variable Variable Variable Total volume 210 pl 84 pl 168 pl A2 Carefully remove the DNA QC Plate from its sealed bag A3 Recommended Dispense the reaction mix into a RT PCR Array Loading Reservoir to assist in loading A4 Add reaction mix to each well of the DNA QC Plate as follows For 96 well plate add 25 ul reaction mix For 384 well plate add 10 ul reaction mix For Rotor Disc 100 add 20 ul reaction mix Note Change pipet tips following each addition to avoid any cross contamination between the wells A5 Carefully but tightly seal the DNA QC Plate with Optical Thin Wall 8 Cap Strips formats A and D Optical Adhesive Film formats C E F and G or Rotor Disc Heat Sealing Film format R qBiomarker Copy Number PCR Handbook 08 2012 45 Remove any air bubbles in any of the plate wells by centrifuging the plate at 1000 rpm for 1 min 96 well plate formats A C D F 2000 rpm for 2 min 384 well plate formats E G This step is not necessary for the Rotor Disc format R A7 Place the DNA QC Plate on ice while setting up the PCR cycling program in step 8 A8 Program the thermal cy
46. ock 384 well block Eppendorf Mastercycler ep realplex models 2 25 4 45 Stratagene models Mx3000P Mx3005P 4000 Takara TP 800 eee 6 qBiomarker Copy Number PCR Handbook 08 2012 qBiomarker SYBR ROX 2 12 24 8 25 ml FAST Mastermix Catalog no 337840 337842 337843 337841 337849 Number of array 2x 12x 24 x 4x 2000 x reactions 96 well 96 well 96 well 384 25 pl well reactions 2x Mastermix 2x 12x 24 x 8 x 25 ml containing HotStart 1 35 1 35 1 35 1 35 ml Taq Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP SYBR Green dye ROX passive reference dye Suitable for use with the Applied Biosystems models 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 with or without ROX filter set Stratagene models Mx3000 Mx3500 Mx4000 Takara TP 800 Rotor Gene QIAGEN Rotor Gene 6000 Shipping and Storage qBiomarker Copy Number PCR Arrays are shipped at ambient temperature on blue ice cold packs or on dry ice depending on the destination and accompanying products For long term storage store qBiomarker Copy Number PCR Arrays at 15 to 30 qBiomarker Copy Number PCR Assays are shipped at ambient temperature but must be stored at 20 on arrival and at 15 to 30 for long
47. odels iQ5 MyiQ MyiQ2 CFX96 CFX384 Stratagene models Mx3000P Mx3005P Mx4000P Eppendorf Mastercycler ep realplex models 2 25 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 25 4 and 45 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 13 Cycling conditions for Roche LightCycler 480 Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 45 15 25 C 1 min 60 C Perform fluorescence data collection 1 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 Table 14 Cycling conditions for Rotor Gene cyclers Cycles Duration Temperature Comments 1 10 min 95 HotStart DNA Taq Polymerase is activated by this heating step 40 15 95 6 30 60 Perform fluorescence data collection qBiomarker Copy Number PCR Handbook 08 2012 37 Table 15 Cycling conditions for Bio Rad and Takara cyclers and other cyclers Cycles
48. on key CNVs known to contribute or correlate with selected complex human diseases genetic diseases or traits Each qBiomarker Copy Number PCR Array enables analysis of up to 95 genes or regions of interest GOls or ROls The arrays are compatible with most real time PCR platforms Copy number assays qBiomarker Copy Number PCR Assays provide a method for obtaining specific accurate reproducible and easy to interpret copy number change results for an individual gene or region of interest or ROI Every assay has been bench verified and is ready to use in microarray follow up studies specific target screening and related studies 10 qBiomarker Copy Number PCR Handbook 08 2012 Principle and procedure All qBiomarker Copy Number PCR Assays are designed in unique regions of the genome A multi copy reference assay the qBiomarker Multicopy Reference Copy Number PCR Assay MRef is included on each array The qBiomarker Multicopy Reference Copy Number PCR Assay is also recommended for use with each qBiomarker Copy Number PCR Assay must be ordered separately cat no 0000000 The reference assay recognizes a stable sequence that appears in the human genome over 40 times and whose copy number is not affected or minimally affected by local genomic changes Inclusion of this reference assay during testing allows use of the AAC method to accurately make copy number calls or relative copy number change calls for specific
49. onsult the appropriate material safety data sheets MSDSs available from the product supplier 40 qBiomarker Copy Number PCR Handbook 08 2012 Comments and suggestions Potentially amplified genes have lower copy number and potentially deleted genes have higher copy number Non recurrent events Copy number loss of large chromosomal segments containing the potentially amplified genes can occur when the genes are unrelated to the tumor growth Similarly copy number gain can occur for tumor suppressor genes Chromosomes or chromosome segments containing tumor suppressor genes that are inactivated by point mutations can also show copy number gain Copy number is lower than expected in heterogeneous samples Non tumor cells in the the presence of non tumor cells that have sample normal diploid genomes the copy number call is dependent on the copy number of the target gene in the cancer cells and the amount of non tumor cells in the heterogeneous sample If the percentage of non tumor cells in the sample can be estimated the copy number for a gene can be estimated based on Table 16 qBiomarker Copy Number PCR Handbook 08 2012 41 Table 16 Evaluating gene copy number for cancer cells in the presence of non tumor cells Expected copy number in sample with indicated percentage of normal cells Gene copy number in cancer cells 12 5 25 50 75 87 5 0 0 25 0 5 1 1 5 1 75 3 2 9 2 8 2 5 253 2 1 4 3 8 3 5 3 0 2 5 2 3 6
50. options for a calibrator sample DNA from a fresh frozen sample can be used as a calibrator sample although the final copy number call may be largely influenced by the quality difference between the test sample and the calibrator sample Alternatively DNA from an FFPE sample can be used as a calibrator sample In this case it is preferable to include multiple calibrator samples in the study Only Copy Number PCR Array Format and qBiomarker SYBR ROX FAST Mastermix should be used with Rotor Gene cyclers Take time to familiarize yourself with the real time PCR cycler before starting the protocol Read the cycler user manual and follow the manufacturer s instructions for proper cycler operation and maintenance The chemically modified hot start enzyme along with other proprietary chemical components in the qBiomarker SYBR Mastermix is essential for successful function of the arrays Do not replace any of these reagents with other commercial counterparts calibrator DNA sample is included it should be run in the same real time PCR cycler as the test samples 30 qBiomarker Copy Number PCR Handbook 08 2012 Pipetting accuracy and precision affects the consistency of results Be sure that all pipets and instruments have been checked and calibrated according to the manufacturer s recommendations Make sure that no bubbles are introduced into the wells of the PCR array during pipetting
51. ormation see also the Frequently Asked Questions page at our Technical Support Center www qgiagen com FAQ FAQList aspx 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 giagen com Comments and suggestions C values very high across all wells for a sample a Cycling program Make sure to program the real time PCR cycler incorrect with the temperature profile shown in the protocols Repeat the PCR with corrected settings if necessary b Poor DNA quality Check the DNA quality using the DNA QC Plate see Appendix A Quality Control of Genomic DNA Using the DNA QC Plate page 43 or on an agarose gel to see if the DNA is degraded If the DNA is not degraded it could be of insufficient purity We recommend using one of the kits indicated in Table 3 page 16 for isolation of high quality DNA c Too little DNA used Make sure that the DNA has been properly quantified During the DNA purification process it is essential to perform an RNase digestion RNA contamination in the DNA sample will lead to overestimation of DNA quantity Note that larger amounts of DNA are recommended when working with FFPE samples When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please c
52. r Copy Number PCR Assays For use of qBiomarker Copy Number PCR Arrays 96 well and 384 well plates see PCR Using qBiomarker Copy Number PCR Array Formats A C D F page 22 For use of qBiomarker Copy Number PCR Arrays in Rotor Discs see PCR Using qBiomarker Copy Number PCR Arrays Format R page 30 Important points before starting 34 Before beginning the procedure read Important Notes pages 16 21 Make sure that the sample cell population contains at least 2596 tumor cells for CNA profiling analysis A calibrator sample is a DNA sample with known copy number for the targets of interest When a calibrator sample or samples are used we recommend that if possible the calibrator sample is from the same tissue as the test sample We also recommend that the DNA quality of the calibrator sample is comparable to that of the test sample see Appendix A Quality Control of Genomic DNA Using the DNA QC Plate page 43 for details of assessment of sample quality For fresh frozen test samples DNA from a fresh frozen sample should also be used as a calibrator sample For FFPE test samples there are 2 options for a calibrator sample DNA from a fresh frozen sample can be used as a calibrator sample although the final copy number call may be largely influenced by the quality difference between the test sample and the calibrator sample Alternatively DNA from an FFPE sample can be used as a calibrator sample In
53. repare a reaction mix according to Table 4 for each sample Recommended amounts of genomic DNA per sample are shown in Table 5 Table 4 Preparation of reaction mix 96 well 384 well 384 well 23 gene 23 gene 95 gene panel panel panel Formats Formats Formats Array format A C D F E G E G qBiomarker SYBR 28 1350 ul 550 ul 2100 ul 0 4 1 0 ug 0 2 0 5 ug 0 8 2 0 ug T fresh fresh fresh 0 8 1 2 0 4 1 0 1 6 4 0 ug FFPE FFPE FFPE Variable Variable Variable water Total volume 2700 ul 11001 4200 This volume provides an excess of 300 ul per sample to allow for some imprecision in pipetting This volume provides an excess of 140 per sample to allow for some imprecision in pipetting This volume provides an excess of 360 per sample to allow for some imprecision in pipetting IRR ame qBiomarker Copy Number Handbook 08 2012 23 Table 5 Recommended amounts of genomic DNA per sample 96 well 384 well 384 well 23 gene 23 gene 95 gene panel panel panel Sample type Figure 1 Figure 2 Figure 3 Fresh tissue 0 4 1 0 ug 0 2 0 5 ug 0 8 2 0 ug FFPE 0 8 1 2 ug 0 4 1 0 ug 1 6 4 0 ug 3 Carefully remove the PCR array plate from its sealed bag 4 Recommended Dispense the reaction mix into a RT PCR Array Loading Reservoir to assist in loading 5 Add reaction mix to e
54. sc 100 format DNA QC Plate can be used for quality control of 12 DNA samples qBiomarker Copy Number PCR Handbook 08 2012 43 pejooipur aduasajos y 96 4 DO WNC 104 1 4 79 4 OdWS OdWS OdWS OdWS OdWS OdWS OdWS OdWS OdWS OdWS OdWS OdWS H Nad 34 34 34 34 34 34 34 34 Nad 34 9 VOEld VOEMId VOEMId VOElld VOElld VOElld VOElld VOElld VOElld amp DI3W 3 SVAN SVAN SVAN SVAN SVAN SVAN SYAN SVAN SVAN SVAN SVAN SYIN 5 5 5 SVadH 5 5 5 SVdH SVaH SVaH 5 SVaH Sve SVJ SVJ S Val S Vl Sve Svea S Val S Val S Vl Sve Svar 8 dvad dvad dvad dvad dvad dvad dvad Avg Jvdd V el LL 01 6 8 4 9 4 V 4 L qBiomarker Copy Number PCR Handbook 08 2012 44 Important points before starting See Important points before starting page 22 before beginning the procedure Procedure a reaction mix according to Table 17 Note Use the qBiomarker Probe Mastermix included with the DNA QC Plate Note It is recommended to prepare a reaction mix for 8 4 replicate PCRs for each sample Prepare more mix than is required so as t
55. term storage qBiomarker SYBR Mastermixes are shipped on blue ice cold packs For long term storage keep tubes at 20 C If the entire volume will not be used at once we recommend dividing into aliquots and storing at 20 Avoid repeated freezing and thawing If stored under these conditions qBiomarker SYBR Mastermixes are stable for 6 months after receipt Intended Use qBiomarker Copy Number PCR Arrays qBiomarker Copy Number PCR Assays and qBiomarker SYBR Mastermixes are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease qBiomarker Copy Number PCR Handbook 08 2012 7 All due and attention should be exercised 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 your expectat
56. this case it is preferable to include multiple calibrator samples in the study Take time to familiarize yourself with the real time PCR cycler before starting the protocol See the cycler user manual and follow the manufacturer s instructions for proper cycler operation and maintenance The chemically modified hot start enzyme along with other proprietary chemical components in the qBiomarker Copy Number Reference Mix and qBiomarker SYBR Mastermix is essential for successful function of the assays Do not replace any of these reagents with other commercial counterparts If a calibrator DNA sample is included it should be run in the same real time PCR cycler as the test samples Pipetting accuracy and precision affects the consistency of results Be sure that all pipets and instruments have been checked and calibrated according to the manufacturer s recommendations qBiomarker Copy Number PCR Handbook 08 2012 Make sure that no bubbles are introduced into the wells of the PCR plate during pipetting Do not use DEPC treated water Use high quality nuclease free water If precipitates are present in the mastermix tube warm the reagents at 42 for 1 min and vortex briefly to dissolve Repeat if necessary Procedure 1 Briefly centrifuge the qBiomarker SYBR Mastermix 10 15 s to bring the contents to the bottom of the tube Note As the qBiomarker SYBR Mastermix contains HotStart DNA Taq Polymerase that is acti
57. ure the average background of each well is determined just before amplification commences Optional Select Ignore First Fluorescent signal from the initial cycles may not be representative of the remainder of the run Thus better results may be achieved if the initial cycles are ignored Up to 5 cycles can be ignored Optional Select Noise Slope Correction Selection of this option can improve data whose baseline initial cycles is noticeably sloped Noise qBiomarker Copy Number PCR Handbook 08 2012 Slope Correction improves the data when raw data backgrounds are observed to slope upward or downward before the takeoff point Note Ensure that the settings are the same across all qBiomarker Copy Number PCR Array runs in the same analysis 9 Manually define the threshold by using the log view of the amplification plots Choose a threshold value above the background signal but within the lower one third to lower half of the linear phase of the amplification plot Note Ensure that the threshold values are the same across all qBiomarker Copy Number PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays 10 Export the C values for all wells to a blank Excel spreadsheet for use with the qBiomarker Copy Number PCR Array Data Analysis Web based software Note qBiomarker Copy Number PCR Array Data Analysis Software is avail
58. ve only after heat activation reactions can be prepared at room temperature 15 25 C 2 Prepare a reaction mix according to Table 11 For genomic DNA from fresh or frozen tissue samples it is recommended to start with 4 ng DNA per well if setting up reactions in 96 well plates or 100 well Rotor Discs or 2 ng DNA per well if setting up reactions in 384 well plates Due to the fragmented nature of DNA extracted from FFPE samples it is recommended to use 8 20 ng DNA per well if setting up reactions in 96 well plates or 100 well Rotor Discs or 4 10 ng DNA per well if setting up reactions in 384 well plates Note It is recommended to prepare a reaction mix for a minimum of 4 replicate reactions for each sample Prepare 596 more mix than is required to allow for imprecision in pipetting qBiomarker Copy Number PCR Handbook 08 2012 35 Table 11 Preparation of reaction mix Format 96 well Rotor Disc 384 well plate plate Number of reactions 4 4 4 qBiomarker SYBR Mastermix 2x SH 29 16 ng fresh 16 ng fresh 8 ng fresh Genomic DNA 32 80 ng 32 80 ng 16 40 ng FFPE FFPE FFPE qBiomarker Copy Number PCR 5 ul 4 ul 2 ul Assay Nuclease free Variable Variable Variable water Total volume 100 pl 80 pl 40 pl Add 25 pl reaction mix to each well of a 96 well plate or 20 pl reaction mix to each well of a Rotor Disc 100 or 10 pl reaction mix to each well of a 384 well plate using a single
59. y qBiomarker Copy Number PCR Assay 200 qBiomarker SYBR Fluor Mastermix 2 qBiomarker SYBR ROX Mastermix 2 qBiomarker SYBR ROX FAST Mastermix 2 Related products DNA QC Plate Nuclease Free Water 10 x 50 ml Nuclease Free Water 1000 ml Nuclease Free Water 5 liters RT PCR Array Loading Reservoir Contents Arrays of assays for real time PCR based copy number alteration and variation profiling available in 96 well 384 well and Rotor Disc formats For 200 reactions Mix of 2 gene specific primers provided in solution For 2 x 96 assays in 96 well plates For 2 x 96 assays in 96 well plates For 2 x 96 assays in 96 well plates Array of assays for evaluating DNA quality for real time PCR based arrays available in 96 well 384 well and Rotor Disc formats 10 x 50 ml nuclease free water prepared without the use of DEPC diethylpyrocarbonate 1000 ml nuclease free water prepared without the use of DEPC diethylpyrocarbonate 5 liters nuclease free water prepared without the use of DEPC diethylpyrocarbonate 12 x 5 ml capacity irradiation sterilized loading reservoirs Cat no Varies 337812 337830 337820 337840 Inquire 129114 129115 129117 338162 QIAGEN reserves the right to occasionally redesign individual qBiomarker Copy Number PCR Assays for improved performance This revision history can be accessed by contacting Technical Services and supply

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