Sample & Assay Technologies EpiTect® Methyl II PCR
Contents
1. 30 ul 300 ul 660 pl M 330 ul 30 ul 300 660 pl Mg M 330 ul 330 30 ul 30 ul 300 ul 300 ul 660 ul 660 pl 2 Mix tubes well by vortexing and briefly centrifuge the contents to the bottom of the tube 3 Add 25 ul of the M reaction to each well in rows A and B of the 96 well EpiTect Methyl II Signature PCR Array Add 25 pl of the M reaction to each well in rows C and D Add 25 ul of the M reaction to each well in rows E and F Finally add 25 pl of the M reaction to each well in rows G and H as shown in Figure 2 Digest Well 1 2 3 4 5 6 7 8 saccadic na es ve enr ne ns ono m en sec oec 9 0 11 12 Fe me on ore 9 coa os coe cos Gos c10 G12 M e13 G14 615 616 617 019 620 621 ez2 sec DEC om en e oon eo er on neve Jon Mg ss es essa e n en em oza see wc e 601 602 coa cos coe co co cro G12 M a e13 e14 615 18 019 620 G21 622 sec pec Figure 2 Layout of a 96 well EpiTect Methyl Il Signature PCR Array EpiTect Methyl II PCR Array Handbook 02 2012 4 Seal or cap the wells of the plate Centrifuge the plate for 1 min at 2000 rpm to remove any air bubbles Running the PCR 1 Program the thermal cycler according to the manufacturer s instructions using the conditions ou2. Mx3000P Mx4000 Stratagene Excel Microsoft Microsoft Corporation 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 EpiTect Methyl Il PCR Array to the following terms 1 The EpiTect Methyl Il PCR Array may be used solely in accordance with the EpiTect Methyl Il PCR Array 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 EpiTect Methyl II PCR Array 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 OT ya 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 investiga
3. qPCR Mastermix 2 suitable for use with the following real time cyclers Bio Rad models iCycler 195 MyiQ MyiQ2 RT PCR Array 5 ml capacity reservoir for convenient 338162 Loading Reservoir sample loading on PCR arrays 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 qiagen com or can be requested trom QIAGEN Technical Services or your local distributor Larger kit sizes available please inquire HR wT SH UT EpiTect Methyl PCR Array Handbook 02 2012 41 Notes 42 EpiTect Methyl II PCR Array Handbook 02 2012 QIAGEN reserves the right to occasionally redesign individual assays on the EpiTect Methyl Il PCR Arrays for improved performance This revision history can be accessed by contacting technical support and providing the batch numbers from your arrays Trademarks QIAGEN AllPrep DNeasy EpiTect RNeasy QIAGEN Group Applied Biosystems StepOnePlus ROX SYBR Life Technologies Corporation Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ MyiQ2 Bio Rad Laboratories Inc Eppendorf Mastercycler Eppendorf AG MethylScreen Orion Genomics LLC Roche LightCycler Roche Group SmartCycler Cepheid Stratagene Mx3005P
4. by gently pipetting up and down c RNA contamination in contamination inhibits restriction enzyme the DNA samples DNA digestion and causes an overestimation of DNA concentration Be sure to include any RNase treatment steps recommended in the procedure of the chosen DNA preparation kit E Other contaminants in DNA prepared from difficult organ tissues may the DNA samples contain protein and or polysaccharide contaminants that significantly inhibit restriction enzyme activity Organic reagents such as chloroform phenol and isopropanol used in some DNA kits and protocols may not be completely removed Be sure to use the recommended DNA isolation kits and protocols and avoid using organic solvent based methods and protocols for DNA preparation e Too much DNA used in Carefully measure the DNA concentration and the digestion only use the amount of DNA recommended by the selected PCR setup protocol EpiTect Methyl II PCR Array Handbook 02 2012 37 Comments and suggestions f Incorrect incubation Incubate for at least 4 hours at 37 C and use the conditions size of tubes recommended in the protocol Use an overnight incubation if a shorter time was used previously and resulted in incomplete digestion High mock digestion M C values from most all genes Insufficient DNA used Be sure to use at least the amount of DNA in the digestion recommended in the protocol Use the recommended methods and instruments to determin
5. convenient and compact PDF format at www qgiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 EpiTect Methyl II PCR Array Handbook 02 2012 7 Introduction The EpiTect Methyl Il PCR System is an innovative technology enabling fast and accurate CpG island DNA methylation profiling of individual genes as well as disease and pathway focused gene panels The technology provides ready to use predesigned primer assays with high specificity and amplification effeciency to detect the methylation status of the promoter region gene of interest Arrays are disease or pathway focused and enable detection of the methylation status of 22 or 94 genes simultaneously EpiTect Methyl Il PCR Arrays use the MethylScreen Technology provided under license from Orion Genomics LLC Approximately 60 70 of all human gene promoters overlap with CoG islands regions with an elevated GC content and a high frequency of CpG dinucleotides Gene silencing by means of DNA methylation of specific gene promoters is a well known feature of neoplastic cells and plays an important role in normal cell differentiation and development 1 DNA methylation occurs mainly at CpG dinucleotides and involves the enzymatic add
6. for use or storage at 20 C Mix the samples thoroughly by vortexing before use Centrifuge the samples down briefly and proceed to step 1 of Setting up the PCR EpiTect Methyl II PCR Array Handbook 02 2012 23 Setting up the PCR 1 Prepare a reaction for each of the 4 digestions M Mg and M a in a 1 5 ml tube according to Table 11 Table 11 PCR setup Component M M M M PCR master mix 590 ul 590 590 ul 590 uul M digest 120 ul M digest 120 ul M digest 120 ul digest 120 ul RNase DNase free water 470 ul 470u 470ul 470 yl Final volume 1180 pl 1180yl 1180 jl 1180 pl 2 Mix tubes well by vortexing and briefly centrifuge the contents to the bottom of the tube 3 Carefully add each reaction mix to the appropriate wells of the EpiTect Methyl 1 Complete PCR Array 384 well plate as follows using the provided 384EZLoad Covers Figure 4 next page Place Cover 1 on the plate Add 10 jl M reaction to the open wells odd number wells of rows A C E G I K M and O Remove and discard the cover Place Cover 2 on the plate Add 10 pl M reaction to the open wells even number wells of rows A C E G I K M and O Remove and discard the cover Place Cover 3 on the plate Add 10 pl M reaction to the open wells odd number wells of rows B D F H J L N and P Remove and discard the cover Place Cover 4 on the plate Add 10 pl M reaction to the
7. is critical that the cycling conditions are followed exactly Table 16 PCR cycling protocol Temperature Time Number of cycles 95 C 10 min 1 cycle 996 30 s P 3 cycles 72 min 97 15s 40 cycles 72 1 mint According to instrument Melting curve segment recommendations Hot start to activate DNA polymerase t Detect and record SYBR Green fluorescence from each well during the annealing step of each cycle 2 After the run has finished analyze the data as described in Data Analysis page 30 EpiTect Methyl PCR Array Handbook 02 2012 29 Data Analysis Obtaining raw threshold cycle C values After the cycling program has completed obtain the values according to the instructions provided by the manvfacturer of the real time PCR instrument We recommend manually setting the baseline and threshold values as follows Note when comparing multiple plates make sure that the settings for all plates are identical Baseline Using the Linear View of the amplification plots set the instrument to use the readings from cycle number 2 through the cycle just before the earliest visible amplification usually between cycle 10 and 15 Threshold value Using the Log View of the amplification plots place the threshold above the background signal but within the lower third of the linear portion of the amplification curves Exporting values Export and or copy paste the values from
8. microcentrifuge 4 Set up 4 digestion reactions M M Mg and M according to Table 2 IMPORTANT All 4 tubes must contain equal amounts of genomic DNA 14 EpiTect Methyl II PCR Array Handbook 02 2012 Table 2 Restriction digestion Component M M M M a Reaction mix from step 3 28 ul 28 ul 26 28yl Methylation sensitive enzyme A E 1 ul 1 ul Methylation dependent enzyme B E 1 ul 1 ul RNase DNase free water 2 ul 1 ul 1 ul Final volume 30 ul 30l 30pl 30 5 Pipet up and down to gently but thoroughly mix the components Centrifuge the tubes briefly in a microcentrifuge IMPORTANT Do not vortex 6 Incubate all 4 tubes at 37 C for 6 h in a heating block or thermal cycler The reaction can also be performed overnight 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 min 8 The reactions are now ready for use or storage at 20 C Mix the samples thoroughly by vortexing before use Centrifuge the samples briefly in a microcentrifuge and proceed to step 1 of Setting up the PCR EpiTect Methyl II PCR Array Handbook 02 2012 15 Setting up the PCR 1 Prepare a reaction for each of the 4 digestions M M Mg and M in a 1 5 ml tube according to Table 3 Table 3 PCR setup Component PCR master mix M digest M digest M digest digest RNase DNase free water Final volume 330 pl
9. the amount in each digest with that of a mock no enzymes added digest using a AC method The reliability and simplicity of the procedure make this technology highly suited for semi high throughput DNA methylation profiling and biomarker development for various research fields such as stem cell differentiation and development 8 EpiTect Methyl II PCR Array Handbook 02 2012 A protocol overview of the EpiTect Methyl Il PCR Array is shown in Figure 1 Briefly input genomic DNA is aliquoted into four equal portions and subjected to mock no enzyme methylation sensitive MSRE methylation dependent MDRE and double MSRE and MDRE restriction endonuclease digestion After digestion the enzyme reactions are mixed directly with qPCR master mix and are dispensed into a PCR Array plate containing pre aliquoted primer mixes Real time PCR is carried out using specified cycling conditions Finally the raw AC values are pasted into the data analysis spreadsheet which automatically calculates the relative amount of methylated and unmethylated DNA fractions y CN 71707 No Methylation Methylation MSRE enzyme sensitive dependent Mo enzyme enzyme MDRE Ms d 4 123456728 9101112 A esc poo exc 999009000 D F EL G H Methylated l E Unmethylated 100 9 40 20 0 Bs Oe oY ow P SSE ZTSSSAES Figure 1 EpiTect Methyl Il PCR pro
10. the instrument software to a blank Microsoft Excel spreadsheet according the manufacturer s instructions for the real time PCR instrument Microsoft Excel based data analysis template First download the EpiTect Methyl Il PCR Array Microsoft Excel based data analysis template which is available at www Sabiosciences com dna methylation data analysis php Then paste in the value data and analyze the automatically generated results by following the directions in the Instructions worksheet of the Excel file Data quality control Mock digest M values The values of the mock digests for all genes should be within the range of 18 to 27 cycles if the recommended amounts of genomic DNA were used Single enzyme digest and values The values of the M and M digests should be between the values of the mock and double digests depending on the methylation status of the DNA samples 30 EpiTect Methyl II PCR Array Handbook 02 2012 Double digest M C values The C values of the double digests should be higher than the C values of the mock digest Enzyme digestion efficiency The difference values between the double digest and mock digest samples represents the analytical window W of the assay and should be greater than 3 AC M gt 3 This means that more than 87 5 of all DNA molecules in the samples were digested and that the assay results are reliable and meaningf
11. to 12 DNA samples 5x Restriction Digestion Buffer Methylation Sensitive Enzyme A Methylation Dependent Enzyme B EpiTect Methyl II PCR Laboratory tested forward and reverse 335002 Assay 200 primers for 200 x 25 ul reactions 25 ul per primer total volume 200 ul DNeasy Blood and 50 DNeasy Mini Spin Columns 69504 Tissue Kit 50 Proteinase K Buffers Collection Tubes 2 ml AllPrep DNA RNA For 50 minipreps AllPrep DNA Spin 80204 Mini Kit 50 Columns RNeasy Mini Spin Columns Collection Tubes RNase Free Water and Buffers Larger kit sizes available please inquire 40 EpiTect Methyl II PCR Array Handbook 02 2012 Product Contents Cat no RT SYBR Green qPCR For 2 x 96 assays in 96 well plates 330500 Mastermix 2 suitable for use with real time cyclers that do not require a reference dye including Bio Rad models CFX96 CFX384 Bio Rad MJ Research Chromo4 Bio Rad MJ Research Opticon 2 Roche LightCycler 480 96 well and 384 well all other cyclers RT SYBR Green ROX For 2 x 96 assays in 96 well plates 330520 qPCR Mastermix 2 suitable for use with the following real time cyclers Applied Biosystems models 5700 7000 7300 7500 Standard and Fast 7700 7900HT 96 well block Standard and Fast and 384 well block StepOnePlus Eppendorf Mastercycler ep realplex models 2 25 4 45 Stratagene models Mx3000P Mx3005P Mx4000 RT SYBR Green Fluor For 2 x 96 assays in 96 well plates 330510
12. values can be used to generate a graphical representation of the data using our developed Hierarchical Clustering method www sabiosciences com dna methylation heatmap php Significance of the level of methylated DNA 96 of total input DNA must be defined by the researcher Methylated DNA may have biological significance if such methylation status is associated with a specific tumor tissue or other phenotype Ideally to determine if this methylation status is sufficient to repress transcription measuring the corresponding expression levels and comparing those with the expression levels in the appropriate controls should be considered Alternatively results can be compared between a control and experimental DNA samples Such parallel analysis will allow researchers to see if the methylation status of an experimental sample is substantially different from a matched control sample i e tumor sample vs normal control or treated sample vs untreated AC data analysis Due to the inversely proportional relationship between threshold cycle and the amount of input DNA and due to the doubling of PCR product with every cycle in the exponential phase of the reaction the initial DNA amount in each digest before PCR is expressed as Cmo 2797 Chs 2 0M 2 0d 2770 1 The fraction of DNA in each digest is calculated by normalizing the DNA amount to the amount of digestible DNA The amount of digestible DNA is equal to the to
13. 02 2012 We recommend the DNeasy Blood and Tissue Kit or the AllPrep DNA RNA Mini Kit for preparation of genomic DNA samples When using the DNeasy Blood and Tissue Kit ensure that samples have been treated for the removal of RNA as RNA contamination will cause inaccuracies in DNA concentration measurements and may affect restriction digestion efficiency Do not omit the recommended RNase treatment step to remove RNA If genomic DNA samples are harvested from biological samples where purification kits are not available contact QIAGEN Technical Services for suggestions For best results resuspend or dilute all DNA samples in DNase free water or alternatively in DNase free 10 mM Tris buffer pH 8 0 without EDTA Measurement of DNA concentration and purity by UV spectrophotometry Prepare dilutions of genomic DNA samples and measure absorbance in DNase free 10 mM Tris buffer pH 8 0 The spectral properties of nucleic acids are highly dependent on pH The recommended ratios and values for DNA are as follows E Ao o A230 gt 7 Ao o A280 gt 8 Aso concentration 74 ug ml DNA concentrations for restriction digestion and PCR assay Using the recommended amount of DNA optimizes the sensitivity of detecting methylated DNA More input DNA may be used if analyzing hypermethylated DNA isolated from samples of heterogeneous cell types e g tumor samples where heavy non tumor cell contamination is expected e g blood stromal
14. 2 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
15. 30519 for Bio Rad iCycler MyiQ MyiQ2 and iQ 5 instruments RT SYBR Green qPCR Mastermix cat nos 330500 330502 330503 330501 330509 for instruments that do not require a reference dye e g Bio Rad models CFX96 CFX384 Bio Rad MJ Research Opticon 2 Bio Rad MJ Research Chromo4 Roche LightCycler 480 96 and 384 well RT PCR Array Loading Reservoirs cat no 338162 Real time PCR instrument see list of supported instruments on page 5 Calibrated single and multi channel pipets RNase DNase free pipet tips and tubes RNase DNase free 100 ul regular PCR tubes 8 or 12 tube strings Molecular biology grade RNase and DNase free water EpiTect Methyl II PCR Array Handbook 02 2012 11 Important Notes Controls for monitoring enzyme digestion efficiency A successful EpiTect Methyl 1 PCR experiment relies on efficient DNA digestion by methylation sensitive and methylation dependent restriction enzymes Each EpiTect Methyl II PCR Array includes specific control assays for monitoring the cutting efficiencies of these enzymes and ensuring reliable and reproducible results After the C values are pasted into the Microsoft Excel data analysis spreadsheet a Pass or Fail result is returned for the SEC methylation sensitive enzyme control and DEC methylation dependent enzyme control assays DNA contamination For reliable results it is very important to prevent contamination of the EpiTect Methyl II PCR assay reaction
16. 35212 A C D F 27 96 well 12 335222 94 96 well 4 3 335222 EG 94 384 well 2 6 22 384 well 4 0 5 12 E Recommended amount for optimal PCR Array performance Array format and real time cycler compatibility Array Suitable real time cyclers Plate format format A Applied Biosystems 5700 7000 7300 7500 96 well Standard 7700 7900HT Standard Bio Rad iCycler iQ 5 MyiQ MyiQ2 Bio Rad MJ Research Chromo4 Eppendorf Mastercycler ep realplex 2 2s 4 4s Stratagene Mx3005P Mx3000P C Applied Biosystems 7500 FAST 7900HT FAST 96 well StepOnePlus D Bio Rad CFX96 Bio Rad MJ Research Opticon 2 96 well Stratagene Mx4000 E Applied Biosystems 7900HT 384 well block Bio Rad 384 well CFX384 F Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 well block 384 well EpiTect Methyl II PCR Array Handbook 02 2012 Storage The EpiTect Methyl Il Signature PCR Array and EpiTect Methyl Il Complete PCR Array are shipped at ambient temperature on ice or on dry ice depending on the destination and accompanying products Upon receipt store at 20 C If stored under these conditions EpiTect Methyl Il Signature PCR Arrays and EpiTect Methyl II Complete PCR Arrays are stable for 6 months after receipt Quality Control In accordance with QIAGEN s Quality Management System each lot of EpiTect Methyl Il Signature PCR Array and EpiTect Methyl Il
17. 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 2
18. Complete PCR Array is tested against predetermined specifications to ensure consistent product quality Product Use Limitations Purchaser agrees that use of this product and data therefrom is limited solely to the purchaser and for only the purchaser s own internal molecular biology research applications Permitted Use and shall not be re sold or used for any other purposes all of which are expressly prohibited including without limitation diagnostic purposes uses that could require regulatory approval for diagnostics from an agency of any government or regulatory entity anywhere in the world diagnosis prevention or treatment of disease and the right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities including without limitation for a fee or other commercial consideration Except for the Permitted Use no rights titles or interests in or to any tangible or intangible property rights are conveyed or shall be deemed conveyed by implication estoppel or otherwise The performance characteristics of the product other than for the Permitted Use are unknown EpiTect Methyl II PCR Arrays 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 guide
19. February 2012 EpiTect Methyl II PCR Array Handbook For pathway or disease focused profiling of regional DNA methylation using MethylScreen technology 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 RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 6 Quality Control 6 Product Use Limitations 6 Technical Assistance 7 Safety Information 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Protocols a EpiTect Methyl Il Signature PCR Array for 22 Genes in a 96 well Format and 1 DNA Sample 14 E EpiTect Methyl 1 Complete PCR Array for 94 Genes 96 well format and 1 DNA Sample 18 a EpiTect Methyl 1 Complete PCR Array for 94 Genes in a 384 well Format and 1 DNA Sample 22 a EpiTect Methyl Il Signature PCR Array for 22 Genes in a 384 well Format and 4 DNA Samples 26 Data Analysis 30 Obtaining raw threshold cycle
20. IAGEN 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 Cited references 1 Esteller M 2007 Epigenetic gene silencing in cancer the DNA hypermethylome Hum Mol Genet 6 R50 2 Ordway JM et al 2006 Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets Carcinogenesis 27 2409 rl EpiTect Methyl II PCR Array Handbook 02 2012 39 Ordering Information Product Contents Cat no EpiTect Methyl II For methylation analysis of 22 genes in a 335212 Signature PCR Array 96 well or 384 well plate format 22 2 12 or 24 x 96 4 x 384 EpiTect Methyl II For methylation analysis of 94 genes in a 335222 Complete PCR Array 96 well or 384 well plate format 94 2 x set of 4 of 96 2 12 or 24 x 384 Related products EpiTect Methyl II For methylation analysis of customer 335112 Custom PCR Array selected genes in a 96 well or 384 well plate format EpiTect Methyl II DNA Reagents for the cleavage of methylated 335452 Restriction Kit 12 and unmethylated DNA for processing up
21. al volume 470 ul Vortex to thoroughly mix the components and centrifuge briefly in a microcentrifuge 4 Setup 4 digestion reactions M M Mg and M according to Table 6 IMPORTANT All 4 tubes must contain equal amounts of genomic DNA 18 EpiTect Methyl II PCR Array Handbook 02 2012 Table 6 Restriction digestion Component M M M M a Reaction mix from step 3 112ul 11241 112 112yl Methylation sensitive enzyme A 4 ul 4 ul Methylation dependent enzyme B 4 ul 4 ul RNase DNase free water 8 ul 4 ul 4 ul Final volume 120 pl 120pl 120pl 120 pl 5 Pipet up and down to gently but thoroughly mix the components Centrifuge the tubes briefly in a microcentrifuge IMPORTANT Do not vortex 6 Incubate all 4 tubes at 37 C for 6 h in a heating block or thermal cycler The reaction can also be performed overnight 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 min 8 The reactions are now ready for use or storage at 20 C Mix the samples thoroughly by vortexing before use Centrifuge the samples briefly and proceed to step 1 of Setting up the PCR Setting up the PCR 1 Prepare a reaction for each of the 4 digestions M M Mg and M in a 15 ml tube according to Table 7 O LLL EpiTect Methyl II PCR Array Handbook 02 2012 19 Table 7 PCR setup Component M M M M PCR master mix 1280u 1280yl 1280ul 1280 ul M digest 120 u
22. cedure EpiTect Methyl PCR Array Handbook 02 2012 Mo Ms Md Msd Isolate DNA Incubate with restriction enzymes Mix remaining DNA with SYBR Green qPCR Mastermix Add to array Real time PCR Analyze data The product of the mock no enzyme digestion represents the total amount of input DNA for real time PCR detection In the methylation sensitive digestion Ms reaction the MSRE will digest unmethylated and partially methylated DNA The remaining hypermethylated DNA DNA in which all CpG sites are methylated will be detected by real time PCR In the methylation dependent digestion Md reaction the MDRE will preferentially digest methylated DNA The remaining unmethylated DNA will be detected by real time PCR In the double digestion Msd reaction both enzymes are present and all DNA molecules both methylated and unmethylated will be digested This reaction measures the background and the fraction of input DNA refractory to enzyme digestion EpiTect Methyl Il PCR Arrays provide gene methylation status as percentage unmethylated UM and percentage methylated M fraction of input DNA Unmethylated represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene Methylated represents fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene Each well of EpiTect Methyl II PCR Array plate contains a differen
23. cells etc However maintain the specific enzyme to DNA ratios outlined below for each assay and purchase additional qPCR plates to ensure assay consistency Using the EpiTect Methyl II DNA Restriction Kit IMPORTANT Do not vortex enzymes Methylation dependent enzyme B is very sensitive to vortexing Extensive vortexing may cause a loss of enzyme activity Mix enzymes by pipetting gently up and down M Store enzymes at 20 C When in use enzymes should be kept on ice EpiTect Methyl II PCR Array Handbook 02 2012 13 Protocol EpiTect Methyl Il Signature PCR Array for 22 Genes in a 96 well Format and 1 DNA Sample Be sure to read Important Notes page 12 before starting the protocol Procedure Restriction digestion 1 Perform the restriction digestions using the EpiTect Methyl II DNA Restriction Kit cat no 335452 2 Prepare a reaction mix without enzymes as indicated in Table 1 We recommend using 1 ug genomic DNA 5x Restriction Digestion Buffer should be thawed and vortexed well before use If any precipitates are present in the buffer continue mixing the buffer until precipitates dissolve Table 1 Reaction mix without enzymes Component Volume Genomic DNA 1 ug Variable 5x Restriction Digestion Buffer 26 ul RNase DNase free water Variable Final volume 120 pl 3 Add RNase DNase free water make the final volume 120 ul Vortex to thoroughly mix the components and centrifuge briefly in a
24. dures In these situations the digest with the greatest difference in value from the mock digest is used to calculate its DNA fraction whether unmethylated or hypermethylated The opposite fraction hypermethylated or unmethylated respectively is instead calculated as one minus the determined fraction The amount of intermediately methylated DNA is then assumed to be negligible Thus the amount of methylated DNA is represented by the hypermethylated DNA fraction If AC M lt 1 0 and AC M M gt 1 0 use following formula to calculate the fraction of hypermethylated DNA 1 Fyn 1 2 2 5 Symbol M M Mg M a R M UM CDH13 19 84 20 60 28 38 30 36 0 068 99 73 0 27 Fm 2 CrM 2 CCrM 2 CrM 0 0027 or 0 27 1 Fun 1 0 0027 0 9973 or 99 73 F 2 Cr M4 C M 2 36 36 19 84 0 068 If AC M M lt 1 0 and AC M gt 1 0 use following formula to calculate the fraction of unmethylated DNA Fc le mbe2 7 e o ere 6 Example Symbol M M M R M UM 22 96 32 73 23 34 40 00 0 0007 0 114 99 886 F F4 722 CM Q CC M 2 C CM 0 00114 or 0 114 Fun 1 1 0 00114 0 99888 or 99 886 F 22 C M4 2 40 00 22 96 0 000796 EpiTect Methyl II PCR Array Handbook 02 2012 35 If both AC M M and AC M M are less than 1 0 then the frac
25. e DNA concentrations a Be sure to include any RNase treatment steps recommended in the procedure of the chosen DNA isolation kit 2 Degraded DNA DNA samples may be contaminated by microbes due to improper storage of DNA samples e g at 4 C Always store DNA samples at 20 C up to 2 years or 80 C indefinitely c PCR array or master Storing PCR array or master mix at inappropriate mix incorrectly stored temperature for extended periods reduces their activity and PCR amplification efficiency Incorrect real time PCR sure to use the correct cycling program cycling program used including 10 minutes at 95 C to fully activate the hot start enzyme in the RT SYBR Green qPCR Mastermix All 4 digests M M Mg M a C values for an individual gene are gt 32 a DNA sample may This may be due to unreported chromosomal contain a different abnormalities insertion or deletions or single sequence relative to the nucleotide polymorphisms SNPs that affect the most recent NCBI EpiTect Methyl Il PCR Assays genome build Verification may require sequencing of the relevant genomic region in the original DNA sample b Homozygous deletions If the C values from all 4 digests for an individual gene but not the majority of genes are 232 genomic homozygous deletion most likely exists at this locus in the genomic DNA of the original sample 38 EpiTect Methyl II PCR Array Handbook 02 2012 References Q
26. eal or cap the wells of each plate Centrifuge the plates for 1 min at 2000 rpm to remove any air bubbles Note One plate can be run immediately and the other 3 plates placed at 20 C until the PCR instrument is ready for another run Do not thaw the plates before running the PCR but place them directly in the PCR instrument Running the PCR 1 Program the thermal cycler according to the manufacturer s instructions using the conditions outlined in Table 8 Note It is critical that the cycling conditions are followed exactly Table 8 PCR cycling protocol Temperature Time Number of cycles 95 C 10 min 1 cycle 99 30 s 3 cycles 72 C min 97 C 15s 40 cycles 72 C 1 mint According to instrument Melting curve segment recommendations Hot start to activate DNA polymerase t Detect and record SYBR Green fluorescence from each well during the annealing step of each cycle 2 After the run has finished analyze the data as described in Data Analysis page 30 EpiTect Methyl II PCR Array Handbook 02 2012 21 Protocol EpiTect Methyl II Complete PCR Array for 94 Genes in a 384 well Format and 1 DNA Sample Be sure to read Important Notes page 12 before starting the protocol Procedure Restriction digestion 1 Perform the restriction digestions using the EpiTect Methyl II DNA Restriction Kit cat no 335452 2 Prepare a reaction mix without enzymes as indicated in Table 9 We recomme
27. ition of a methyl group to the cytosine residue without changing the primary DNA sequences Such modifications at regulatory regions in particular gene promoters correlate well with the transcriptional state of a gene DNA methylation represses transcription while DNA unmethylation can lead to increased transcription levels DNA methylation is an essential mechanism for normal cellular development imprinting X chromosome inactivation and maintaining tissue specificity It can also contribute significantly to the progression of various human diseases The profiling of tumor suppressor genes and other key genes allows the correlation of CpG island methylation status with transcriptional status biological phenotypes or disease outcomes Therefore the results can provide insights into the molecular mechanisms and biological pathways and aid in the discovery and development of biomarkers Principle and procedure The method employed by the EpiTect Methyl Il PCR System is based on detection of remaining input DNA after cleavage with a methylation sensitive and or a methylation dependent restriction enzyme 2 These enzymes will digest unmethylated and methylated DNA respectively Following digestion the remaining DNA in each individual enzyme reaction is quantified by real time PCR using primers that flank a promoter gene region of interest The relative fractions of methylated and unmethylated DNA are subsequently determined by comparing
28. l M digest 120 ul E M digest E 120 ul digest 120 ul RNase DNase free water 1160u 1160ul 1160gl 1160 ul Final volume 2560 ul 2560 pl 2560 pl 2560 pl 2 Mix tubes well by vortexing and briefly centrifuge the contents to the 3 Digest bottom of the tube Add 25 pl of the M reaction to each well in rows A and B of the four 96 well EpiTect Methyl II Complete PCR Arrays Add 25 pl of the M reaction to each well in rows C and D Add 25 ul of the M reaction to each well in rows E F Finally add 25 pl of the M reaction to each well in rows G and H as shown in Figure 3 Well 1 2 3 4 5 6 7 8 9 10 11 12 A oos cos coe zis ei ets is ty ers cri e age eon evs ere ens es eer ens few ERIT gt ene 619 620 a 622 sec nec t 601 ons a s NER te 601 coz coa cos ee a fe En ez e an Figure 3 Layout of 96 well EpiTect Methyl II Complete PCR Array The first of four 96 well plates is laid out as shown On the remaining 3 plates the SEC and DEC assays are replaced by gene specific primers G45 G69 G93 and G46 G70 G94 respectively 20 EpiTect Methyl II PCR Array Handbook 02 2012 4 S
29. lines that have been developed for recombinant DNA experiments or to other applicable guidelines 6 EpiTect Methyl II PCR Array Handbook 02 2012 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 the EpiTect Methyl Il Signature Array EpiTect Methyl II Complete PCR Array or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in
30. move and discard the cover Place Cover 2 on the plate Add 10 pl M reaction to the open even numbered wells of rows A and C for sample 1 rows E and G for sample 2 rows and K for sample 3 and rows M and O for sample 4 Remove and discard the cover Place Cover on the plate Add 10 pl M reaction to the open odd numbered wells of rows B and D for sample 1 rows F and H for sample 2 rows J and L for sample 3 and rows N and P for sample 4 Remove and discard the cover Place Cover 4 on the plate Add 10 pl M reaction to the open even numbered wells of rows B and D for sample 1 rows F and H for sample 2 rows J and L for sample 3 and rows N and P for sample 4 Remove and discard the cover 28 EpiTect Methyl II PCR Array Handbook 02 2012 Well 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 23 24 i Me M 60 607 09 610 Md iMsd Md Msd Md Msd Md Ma Med 8 05 ir ii bo ein GH Ms mr Med Md Msd Md EM p mit Med Md Med i 2 a gt Figure 5 Layout of 384 well Il EpiTect Methyl I Signature PCR Array 4 Seal or cap the wells of the plate Centrifuge the plate for 1 min at 2000 rpm to remove any air bubbles Running the PCR 1 Program the thermal cycler according to the manufacturer s instructions using the conditions outlined in Table 16 Note It
31. nd using 2 Ug genomic DNA The 5x Restriction Digestion Buffer should be thawed and vortexed well before use If any precipitates are present in the buffer continue mixing the buffer until precipitates dissolve Table 9 Reaction mix without enzymes Component Volume Genomic DNA 2 ug Variable 5x Restriction Digestion Buffer 100 ul RNase DNase free water Variable Final volume 470 3 Add RNase DNase free water to make the final volume 470 ul Vortex to thoroughly mix the components and centrifuge briefly in a microcentrifuge 4 Set up 4 digestion reactions M Mg M a according to Table 10 IMPORTANT All 4 tubes must contain equal amounts of genomic DNA SSS S ZZCUXI 22 EpiTect Methyl II PCR Array Handbook 02 2012 Table 10 Restriction digestion Component M M M M Reaction mix from step 3 1l u 116 1l gl 116 Methylation sensitive enzyme A 2 ul 2 Methylation dependent enzyme 2 ul 2 ul RNase DNase free water 4 ul 2 ul 2 ul Final volume 120 pl 120pl 120pl 120 pl 5 Pipet up and down to gently but thoroughly mix the components Centrifuge the tubes briefly in a microcentrifuge IMPORTANT Do not vortex 6 Incubate all 4 tubes at 37 C for 6 h in a heating block or thermal cycler The reaction can also be performed overnight 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 min 8 The reactions are now ready
32. ociation curve for each well in each plate using the instrument s software A single well defined peak should appear in each well If your instrument does not have a default melting curve program run the following program instead 97 C 1 min 55 C 30 sec OPTICS OFF 55 97 at 2 C s OPTICS ON Data Interpretation EpiTect Methyl Il PCR Arrays provide gene methylation status as percentage unmethylated UM and percentage methylated M fraction of input DNA UM represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene M represents the fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene The number of CpG sites methylated in a targeted region can vary within the fraction of methylated input DNA Unmethylated UM Methylated M Example Input genomic DNA Figure 6 Pictorial explanations of results 32 EpiTect Methyl II PCR Array Handbook 02 2012 In the figure above each horizontal bar represents the targeted region of a gene from one genome Biological samples usually contain many genomes derived from many cell types For simplicity five such genomes are depicted here Light and dark circles represent unmethylated and methylated CpG sites respectively The Results worksheet displays the relative percentage of methylated M and unmethylated UM DNA in each target genomic DNA sequence The M
33. open wells even number wells of rows B D F H J L N and P Remove and discard the cover 24 EpiTect Methyl II PCR Array Handbook 02 2012 Well 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M vO Z Zr Figure 4 Layout ol 384 well EpiTect Methyl II Complete PCR Array 4 Seal or cap the wells of the plate Centrifuge the plate for 1 min at 2000 rpm to remove any air bubbles Running the PCR 1 Program the thermal cycler according to the manufacturer s instructions using the conditions outlined in Table 12 Note It is critical that the cycling conditions are followed exactly Table 12 PCR cycling protocol Temperature Time Number of cycles 95 10 min 1 cycle 29C 30 s 3 cycles TC min 97 15 40 cycles 72 C 1 mint According to instrument Melting curve segment recommendations Hot start to activate DNA polymerase t Detect and record SYBR Green fluorescence from each well during the annealing step of each cycle 2 After the run has finished analyze the data as described in Data Analysis page 30 EpiTect Methyl PCR Array Handbook 02 2012 25 Protocol EpiTect Methyl Il Signature PCR Array for 22 Genes in a 384 well Format and 4 DNA Samples Be sure to read Important Notes page 12 before starting the protocol Procedure Restriction diges
34. s with foreign DNA Even very small amounts of foreign DNA can artificially inflate SYBR Green signals yielding false positive results The most common source of contamination in the PCR reagents comes from the products of previous PCR experiments in your working area To minimize contamination follow the recommendations below Wear gloves throughout the entire procedure Use only fresh PCR grade reagents and labware Physically separate the workspace for PCR setup and post PCR work Never open PCR plates or tubes after real time PCR instrument runs Before setting up an experiment decontaminate the PCR workspace and labware pipet barrels tube racks etc with 10 bleach and UV light Preferentially set up reactions in a PCR workstation E Do not remove the protective film from the PCR array until immediately before use Close all tubes containing PCR products as soon as possible after use Treat any labware tips or tubes containing PCR products or other DNA with 1096 bleach before discarding Genomic DNA preparation High quality DNA is a prerequisite for a successful EpiTect Methyl Il PCR assay reaction Therefore sample handling and genomic DNA isolation procedures are crucial to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the DNA or decrease the restriction enzyme activities necessary for optimal DNA digestion 12 EpiTect Methyl II PCR Array Handbook
35. t primer assay mixed with an inert dye This dye does not affect assay performance or fluorescence detection 10 EpiTect Methyl II PCR Array Handbook 02 2012 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier DNeaosy Blood and Tissue Kit cat nos 69504 or 69506 or the AllPrep DNA RNA Mini Kit cat no 80204 for preparation of DNA EpiTect Methyl II DNA Restriction Kit cat 335452 IMPORTANT The EpiTect Methyl II DNA Restriction Kit contains all necessary components for cleavage of methylated and unmethylated DNA and is essential for a complete and successful experiment The reagents included in the kit are sufficient for processing up to 12 Ug of genomic DNA The actual number of samples processed is dependent on the number of genes analyzed and the plate format selected Appropriate RT SYBR Green qPCR Mastermix be sure to select the correct format for the PCR instrument and size and quantity for the number of samples RT SYBR Green ROX qPCR Mastermix cat nos 330520 330522 330523 330521 330529 for Applied Biosystems Stratagene and Eppendorf Mastercycler ep realplex instruments with a ROX filter set RT SYBR Green Fluor qPCR Mastermix cat nos 330510 330512 330513 330511 3
36. tal amount of DNA determined from the mock digest minus the amount of DNA resistant to DNA digestion determined from the double digest Unmethylated UM DNA fraction 2 909 DOP TD EpiTect Methyl II PCR Array Handbook 02 2012 33 Hypermethylated HM DNA fraction DOM S Fim PEE RR Cuo Cua 2989 Dod Intermediately methylated IM DNA fraction 1 Fum Methylated M DNA fraction Fu Fam Fim 3 DNA copies resistant R to enzyme digestion 2 p QACr Mag CrMo 2 ACr Msa Mo C 2 6 Example Symbol M M M M R HM UM M CCNA 23 16 27 11 24 89 36 51 0 0095 6 47 30 15 69 85 2 Cr Ma 2 Cr M 2 C M4 0 3015 or 30 15 Fum 2 CLM 2 CC M 2 C Cr Ma 0 0647 or 6 47 1 Fi Fum 1 0 0647 0 3015 0 6338 or 63 38 Fum Fim 0 0647 0 6338 0 6985 or 69 85 Fe 2 C Ma Cr Mj 2 36 51 23 16 0 009596 _ _ U aL 34 EpiTect Methyl II PCR Array Handbook 02 2012 Methylation sensitive or methylation dependent digest values within one cycle of the mock digest cannot be reliably used to calculate the percentage of either respective methylated DNA fraction Differences in threshold cycles less than one 1 are within the standard error associated with real time PCR instruments and experimental proce
37. tion 1 Perform the restriction digestions using the EpiTect Methyl II DNA Restriction Kit cat no 335452 2 Prepare a reaction mix without enzymes as indicated in Table 13 We recommend using 0 5 ug genomic DNA per sample The amounts shown in Table 13 are for one sample Repeat this process for each sample 5x Restriction Digestion Buffer should be thawed and vortexed well before use If any precipitates are present in the buffer continue mixing the buffer until precipitates dissolve Table 13 Reaction mix without enzymes Component Volume Genomic DNA 0 5 ug Variable 5x Restriction Digestion Buffer 26 ul RNase DNase free water Variable Final volume 125 3 Add RNase DNase free water to make the final volume 125 yl Vortex to thoroughly mix the components and centrifuge briefly in a microcentrifuge 4 Set up 4 digestion reactions M M Mg and for each sample according to Table 14 IMPORTANT All 4 tubes must contain equal amounts of genomic DNA 26 EpiTect Methyl II PCR Array Handbook 02 2012 Table 14 Restriction digestion Component M M M M Reaction mix from step 3 28 ul 28 ul 28 ul 28 ul Methylation sensitive enzyme A 1 ul 1 ul Methylation dependent enzyme B E 1 ul 1 ul RNase DNase free water 2 ul 1 ul 1 ul Final volume 30 ul 30l 30pl 30 5 Pipet up and down to gently but thoroughly mix the components Centrifuge the tubes briefly in a microcentrif
38. tion of both hypermethylated and unmethylated DNA is assigned as 5096 while again the amount of intermediately methylated DNA is negligible Thus the amount of methylated DNA is represented by the hypermethylated DNA fraction Example Symbol M M M M R M UM SFN 24 03 24 03 24 59 40 00 0 001696 50 096 50 096 Fa Frm 50 0 Fe 2 C My C M 2 40 00 24 03 0 0016 AAAAAAAAZAAAZAAAa AA a aN 36 EpiTect Methyl II PCR Array Handbook 02 2012 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qiagen 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 Incomplete restriction enzyme digestion C M a M lt 3 or SEC or DEC Fail in the data analysis worksheet a b Poor quality DNA Repeat the experiment with new DNA samples Low restriction enzyme Check that the EpiTect Methyl II DNA Restriction activity Kit has not expired Be sure to use the correct amount of both enzymes recommended in the protocol for the amount of DNA used Be sure to mix the enzyme
39. tive 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 The EpiTect Methyl Il PCR Arrays use MethylScreen technology provided under license from Orion Genomics LLC This product is compatible for use in real time PCR methodologies including 5 nuclease processes and dsDNA binding dye processes claimed in patents owned by Roche Molecular Systems Inc F Hoffmann La Roche Ltd and Applera Corporation No right to perform any of these patented processes is conveyed expressly by implication or by estoppel to the purchaser by the purchase of this product A license to use these patented processes for the purchaser s own internal research accompanies the purchase of certain reagents of Applied Biosystems and other licensed suppliers or is available from Applied Biosystems 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 2012 QIAGEN all rights reserved 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 55 11 5079
40. tlined in Table 4 Note It is critical that the cycling conditions are followed exactly Table 4 PCR cycling protocol Temperature Time Number of cycles 95 C 10 min 1 cycle 99 C 30 s 3 cycles 72 C min 97 15 40 cycles 72 C 1 mint According to instrument Melting curve segment recommendations Hot start to activate DNA polymerase t Detect and record SYBR Green fluorescence from each well during the annealing step of each cycle 2 After the run has finished analyze the data as described in Data Analysis page 30 EpiTect Methyl II PCR Array Handbook 02 2012 17 Protocol EpiTect Methyl II Complete PCR Array for 94 Genes in a 96 well format and 1 DNA Sample Be sure to read Important Notes page 12 before starting the protocol Procedure Restriction digestion 1 Perform the restriction digestions using the EpiTect Methyl II DNA Restriction Kit cat no 335452 2 Prepare a reaction mix without enzymes as indicated in Table 5 We recommend using 4 Ug genomic DNA 5x Restriction Digestion Buffer should be thawed and vortexed well before use If any precipitates are present in the buffer continue mixing the buffer until precipitates dissolve Table 5 Reaction mix without enzymes Component Volume Genomic DNA 4 ug Variable 5x Restriction Digestion Buffer 100 ul RNase DNase free water Variable Final volume 470 pl 3 Add RNase DNase free water to make the fin
41. uge IMPORTANT Do not vortex 6 Incubate all 4 tubes at 37 C for 6 h in a heating block or thermal cycler The reaction can also be performed overnight 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 min 8 The reactions are now ready for use or storage at 20 C Mix the samples thoroughly by vortexing before use Spin the samples down briefly and proceed to step 1 of Setting up the PCR el EpiTect Methyl II PCR Array Handbook 02 2012 27 Setting up the PCR 1 Prepare a reaction for each of the 4 digestions M Mg and M a in a 1 5 ml tube according to Table 15 The volumes shown in Table 15 are for one sample Table 15 PCR setup Component M M M M PCR master mix 170 ul 170 ul 170u 170 M digest 30 ul M digest 30 ul M digest 30 ul digest 30 ul RNase DNase free water 140 ul 140 ul 140ul 140 yl Final volume 340 pl 340 ul 340 1 340 pl 2 Mix tubes well by vortexing and briefly centrifuge the contents to the bottom of the tube 3 Carefully add each reaction mix to the appropriate wells of the EpiTect Methyl Il Signature PCR Array 384 well plate as follows using the provided 384EZLoad Covers Figure 4 next page Place Cover 1 on the plate Add 10 pl M reaction to the open odd numbered wells of rows A and C for sample 1 rows E and G for sample 2 rows and K for sample 3 and rows M and O for sample 4 Re
42. ul See also the Data QC Report worksheet in the Microsoft Excel data analysis template For every gene the analytical window W values should be 73 and the Fp values should be 12 596 When using the EpiTect Methyl Il PCR Assay for EP SEC cat no EPHS115450 1A and EP DEC cat no EPHS115451 1A to test the cutting efficiencies of the restriction enzymes copy and paste the values for these controls into the Microsoft Excel data analysis template For the SEC assay the difference in Cj values between the methylation sensitive and mock digests should be equal to or greater than 4 AC M M 4 to Pass the quality control Likewise for the DEC control assay the difference in values between the methylation dependent and mock digests should be equal to or greater than 4 AC M M 4 to Pass the quality control Pass means that more than 93 6 of control DNA molecules spiked in buffer 5x Restriction Digestion Buffer were digested showing that the restriction enzymes were active and digested the DNA efficiently See the Result worksheet in the Microsoft Excel data analysis template Both the SEC and DEC assays should show Pass in the analysis Completion digestion ACt Msd Mo EpiTect Methyl II PCR Array Handbook 02 2012 31 Dissociation melting curve Perform the default melting curve program on the instrument immediately after the cycling program Generate the first derivative diss
43. values 30 Exporting C values 30 Microsoft Excel based data analysis template 30 Data quality control 30 Data Interpretation 32 AC data analysis 33 Troubleshooting Guide 37 References 39 Ordering Information 40 EpiTect Methyl II PCR Array Handbook 02 2012 3 Kit Contents EpiTect Methyl Il Signature PCR Arrays Catalog no 335212 Format A C D E F G 96 well plate containing 2 12 2 12 2 12 2 12 lyophilized assays or24 or24 or 24 E or 24 7 384 well plate containing lyophilized assays E 1 2 Optical thin wall 8 cap 24 24 strips 12 per plate 144 144 288 288 Optical adhesive film 212 7 2 12 4 1 plate 24 7 or 24 384EZLoad Covers set of _ _ _ _ 4 4 covers per plate EpiTect Methyl 1 Complete PCR Arrays Catalog no 335222 Format A C D E F G 96 well plate containing lyophilized assays 8 8 8 B 384 well plate containing 2125 lyophilized assays 2 2 or 24 2 or 24 Optical thin wall 8 cap strips 12 per plate d 7 Ie i i Optical adhesive film 8 2 2 12 8 2 12 1 per plate or 24 or 24 384EZLoad Covers set of 2 12 2 12 4 covers per plate E 7 i or 24 7 or 24 4 EpiTect Methyl II PCR Array Handbook 02 2012 EpiTect Methyl Il PCR Array specifications Number of Number of Cat no Number of DNA Hg gDNA DNA and array genes per Plate samples per samples format plate format plate sample per kit 3
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