User Manual - Biomol GmbH
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1. 72 C 1 minute Heiko cine sedmeni According to instrument 9 9 recommendations The 10 minutes step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle B 1 14 After the run the data can be analyzed as described in section V B 2 Using a 96 genes 96 well PCR Array 4 and one DNA sample Restriction digestion B 2 1 Perform the restriction digestions with the Methyl Profiler Enzyme Kit Catalog MeA 03 B 2 2 Prepare a reaction cocktail without enzymes as indicated below For this protocol it is recommended to use 4 ug genomic DNA The 5X digestion buffer should be thawed and vortexed well before use If any precipitates are present in the buffer make sure to continue mixing the buffer until precipitates dissolves RNase DNase free H20 5X Digestion Buffer 100 ul Genomic DNA 4 ug Final cocktail volume 470 ul B 2 3 After adding H2O to make the final cocktail volume 470 ul vortex to thoroughly mix the components and spin down briefly B 2 4 Set up four digestion reactions Mo Ms Md and Msd according to the following table All four tubes must contain equal amounts of genomic DNA Technical Support 888 503 3187 US 301 682 9200 15 Methyl Profiler DNA Methylation PCR Array System Mo Cocktail from previous step B 2 5 Pipette up and down to thoroughly yet gently mix the components S2. B is very sensitive to vortexing Extensive vortexing can cause loss of enzyme activity Instead mix enzymes by gently pipetting up and down e Store enzymes at 20 C When not at 20 C enzymes should be kept on ice gDNA per gDNA per Catalog Genes gree aan eit P aad dis ans be DF 24 96 well 1 1 1 0 25 10 OAOT 96 96 well 4 1 4 1 10 eG 96 384 well 1 1 2 0 5 4 SOE ne 24 384 well 1 4 0 5 0 125 4 The table outlines the recommended genomic DNA input amounts associated with each Methyl Profiler Assay format columns 1 3 In addition the amount of genomic DNA for each individual digestion Mo Ms Md Msd reaction is listed as well as the amount of genomic DNA PCR template that this translates to in the qPCR step If more input DNA Technical Support 888 503 3187 US 11 301 682 9200 Methyl Profiler DNA Methylation PCR Array System per assay is desired please order additional assays amount for each of the four samples B How to set up a Methyl Profiler Assay Four protocols are provided to assist you in setting up the restriction enzyme reactions and PCR assays The setup is dependent on the PCR Array format used in the experiment Protocol Catalog Genes Diecut Sonic Page B 1 bene Be 24 96 well 1 12 B 2 ue 96 96 well 1 15 B 3 pate 96 384 well 1 17 B 4 aa lt 24 384 well 4 20 B 1 Using
3. and Msa C Values for an Individual Gene gt 32 Your DNA sample may contain a different sequence relative to the most recent NCBI Genome Build due to unreported chromosomal abnormalities insertion or deletions or single nucleotide polymorphisms SNPs that affect the Methyl Profiler PCR Assays Verification of this situation may require sequencing of the relevant genomic region in your original DNA sample Another reason is a Homozygous Deletions If the C values from all the four digests for an individual gene but not the majority of genes are equal to or greater than 32 genomic homozygous deletion most likely exists at this locus in the genomic DNA of your original sample Technical Support support sabiosciences com www sabiosciences com 30 Version 2 1 B Frequently Asked Questions Comments 1 How can be sure that the An excess of each restriction enzyme in restriction enzyme digestion is combination with a long incubation time assures complete a complete digestion when the recommended DNA amounts are used The larger the difference between the C values of the mock and double digests W AC M C Mgq the more complete the restriction enzyme digestion Because C values are inversely and exponentially related to the initial amount of DNA material each unit of cycle difference between these digests represents an additional two fold difference in the amount of DNA For example a five cycle difference mea
4. are added in the double digest 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 Tora Input SMA CI 2 Real Time PCR Specific cycling conditions for many real time PCR instruments are indicated in the manual 3 Data Analysis The relative amount of each DNA fraction methylated intermediate methylated and unmethylated is calculated using a standard AC method normalizing the amount of DNA in Figure 1 Methyl Profiler DNA each digest against the total amount of input Methylation PCR Array Protocol Overview DNA in the M digest Technical Support support sabiosciences com www sabiosciences com 6 Version 2 1 Il Materials Provided The PCR arrays are available in five different plate formats each tailored to a specific subset of real time PCR instrument and associated blocks Formats A C D and F are 96 well plates while Formats E and G are 384 well plates Format For Real Time Instrument Plate All ABI standard blocks 7000 7300 7500 7700 7900 A Bio Rad iCycler MyiQ 96 well Bio Rad Chromo 4 MJ Research Stratagene Mx3005p Mx3000p ABI 7500 and 7900HT FAST 96 well blocks c ABI StepOne plus PPOR Bio Rad CFX96 D Bio Rad Opticon and Opticon 2 MJ Research 96 well Stratagene Mx4000 E ABI 7900HT FAST 384 well block 384 well Roche Light
5. cleavage with a methylation sensitive and or a methylation dependent restriction enzyme 3 These enzymes will respectively digest unmethylated and methylated DNA Following digestion the remaining DNA is quantified by real time PCR in each individual enzyme reaction using primers that flank a promoter gene region of interest The relative fractions of hyper methylated intermediate methylated and unmethylated DNA are subsequently determined by comparing the amount in each digest with that of a mock no enzymes added digest The reliability and simplicity of the procedure make this technology an ideal tool for semi high throughput DNA methylation profiling and biomarker development for various research fields like stem cell differentiation and development cancer and other human diseases Simple enzyme digestion Real Time PCR Methyl Profiler DNA methylation data highly comparable to bisulfite sequencing data Technical Support support sabiosciences com www SABiosciences com 4 Version 2 1 References 1 2 Esteller M 2007 Epigenetic gene silencing in cancer the DNA hypermethylome Hum Mol Genet 6 1 R50 59 Weber M ef al 2007 Distribution silencing potential and evolutionary impact of promoter DNA methylation in the human genome Nat Genet 39 4 457 466 Ordway JM et al 2006 Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets Carcinogenesis 27 12 2409 2423 Bene
6. or disease outcomes Therefore the results can provide insights into the molecular mechanisms and biological pathways critical for disease development and aid in the discovery and development of biomarkers DNA methylation detection technologies based on the bisulfite conversion of DNA samples such as bisulfite sequencing and methylation specific PCR related methods have greatly advanced the DNA methylation studies by providing scientists with the ability to analyze the methylome at single base resolution However these technologies have several limitations that involve time constraints bisulfite modification amplification cloning and sequencing suboptimal DNA conversion yields challenging primer design and PCR optimization and low gene and sample throughput In addition most biological significant changes in DNA methylation are known to occur at multiple CpG dinucleotides simultaneously rather than at single bases suggesting that a regional analysis can be representative for the methylation level of a CpG Island within a promoter region gene of interest 2 The Methyl Profiler DNA Methylation PCR System is an innovative technology enabling fast and accurate detection of CpG island DNA methylation profiles of individual genes as well as disease or pathway focused gene panels This technology is an excellent alternative to low throughput bisulfite based methods In brief the method is based on the detection of remaining input DNA after
7. patents require a separate license from Roche Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Drive Foster City California 94404 USA 3 Methyl Profiler DNA Methylation PCR Array System Background and Introduction Approximately 60 to 70 of all human gene promoters overlap with CpG islands which are regions with an elevated GC content and a high frequency of CpG dinucleotides Gene silencing by means of hyper methylation of specific genes promoters is a well known feature of neoplastic cells and also plays an important role in normal cell differentiation and development 1 DNA methylation occurs mainly at CpG dinucleotides and involves the enzymatic addition 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 hyper methylation represses transcription while hypo methylation can lead to increased transcription levels DNA methylation is an essential mechanism for normal cellular development imprinting X chromosome inactivation maintaining tissue specificity and can contribute significantly to the progression of various human diseases The profiling of tumor suppressor genes and other key genes will allow the correlation of CpG island methylation status with transcriptional status biological phenotypes
8. peak and consistently high amplification efficiencies Technical Support 888 503 3187 US 301 682 9200 31 Methyl Profiler DNA Methylation PCR Array System Comments 4 Do need an internal methylated No For any given target sequence the same DNA reference primer pair is used to amplify the four different digests allowing a direct and reliable comparison In contrast bisulfite modification based PCR methods use two different pairs of primers to amplify either methylated or unmethylated templates of a given target sequence after conversion Differences in their amplification efficiencies cause biased results requiring normalization to in vitro methylated reference DNA However its primers must also have similar amplification efficiencies and its methylation must be consistently complete ideals only achieved after careful trial and error based optimization and additional cost 5 Does this method detect No This method examines the methylation methylation at specific CpG status across a CpG rich sequence Other dinucleotides methods only analyze one or two CpG sites in one assay and assume that their methylation status represents the status of the surrounding target sequence These methods include Southern blot MSRE and RLGS and bisulfite conversion based methods like MSP TaqMan based MethyLight MS SNuPE GoldenGate DNA Methylation BeadArray Infinium DNA Methylation BeadChip and COBRA 6 Can I reliably detect
9. values between the double and mock digests should be greater than two ACt Msa Mo gt 2 and represent the analytical window W of the assay When W is greater than 2 it means that more that 75 of all DNA molecules in the samples were digested hence the results are reliable and meaningful See also the Data QC Report worksheet in the Excel data analysis template For each and every gene the analytical window W values should be greater than 2 and the R values should be less than 25 120 99 100 e ys 35 ee 98 2 Pid 96 5 x 75 c 7 2 2 50 2 Q 20 Fj V ACt Msd Mo e Dissociation Melting Curve Perform the default melting curve program on your instrument immediately after the cycling program Generate the first derivative dissociation curve for each well in each plate using your instrument s software A single well define peak should appear in each well If your instrument does not have a default melting curve program run following program instead 95 C 1 min 65 C 2 min OPTICS OFF 65 C to 95 C at 2 C sec OPTICS ON 4 Data Analysis and Interpretation a Relative Amounts of DNA in each Methylated Fraction The Results worksheet displays the relative percentage of hyper methylated HM intermediately methylated IM and unmethylated UM DNA in each target genomic DNA sequence The HM values can be used to generate a graphical Technical Support support sabiosci
10. After loading each plate carefully seal the plate or cap the wells Centrifuge the plates briefly to remove air bubbles at 1000 rpm for 1 minute One plate can be run immediately and the other three plates can be place at 20 C until your 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 Reactions B 2 13 To run the reactions use the two step program shown below for all cyclers Note the unique cycling conditions necessary to ensure proper performance of the assay 97 C 40 72 C Melting curve segment According to instrument recommendations The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle B 2 14 After the run the data can be analyzed as described in section V Technical Support 888 503 3187 US 301 682 9200 17 Methyl Profiler DNA Methylation PCR Array System B 3 Using a 96 genes 384 well PCR Array and one DNA sample Restriction digestion B 3 1 Perform the restriction digestions with the Methyl Profiler Enzyme Kit Catalog MeA 03 B 3 2 Prepare a reaction cocktail without enzymes as indicated below For this protocol it is recommended to use 2 ug genomic DNA The 5X digestion buffer should be thawed and vortexed well before use If any precipitates are present in the buffe
11. Arrays and Master Mixes Combinations see below Version 2 1 PCR Arrays Master Mix Catalog Pack Size Catalog Quantity 2 PA 01 1 1 MeA H M 0 1A C D F 12 PA 01 12 1 24 PA 01 24 1 MeA H M 0 1E G 4 PA 01 8 1 MeA H M 80 0A C D F 8 PA 01 4 2 PA 01 1 MeA H M 30 0E G 12 PA 01 12 1 24 PA 01 24 1 D Additional Equipment and Reagents 1 2 Real Time PCR Instrument Calibrated single and multi channel pipettes RNase DNase free pipette tips and tubes RNase DNase free 100 uL regular PCR reaction tubes 8 or 12 tube strings Molecular biology grade RNase and DNase free H2O Technical Support 888 503 3187 US 9 301 682 9200 Methyl Profiler DNA Methylation PCR Array System IV Protocol A 1 IMPORTANT Please read through this entire protocol before beginning your experiment Tips for a Successful Assay Avoiding DNA contamination For reliable results it is very important to prevent ANY contamination of the Methyl Profiler Assay reactions 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 Please follow the recommendations outlined below Wear gloves throughout the entire procedure Use only fresh PCR grade reage
12. Cycler 480 96 well block 96 well Roche LightCycler 480 384 well block 384 well NOTE The format of a PCR Array is designated by the last letter of the catalog number Before starting any experiment confirm that you have the correct PCR Array format for your instrument 1 Signature 24 Gene Panel 96 Well PCR Arrays MeA H M 0 1A C D and F are available and shipped in sets of two 2 twelve 12 or twenty four 24 2 Signature 24 Gene Panel 384 Well PCR Arrays MeA H M 0 1E G are available and shipped in sets of four 4 3 Complete 96 Gene Panel 96 Well PCR Arrays MeA H M 80 0A C D F are available and shipped as a duplicate set of four different 24 gene 96 well PCR Arrays 4 Complete 96 Gene Panel 384 Well PCR Arrays MeA H M 30 0E G are available and shipped in sets of two 2 twelve 12 or twenty four 24 Each PCR Array shipment includes the arrays and either twelve 12 optical thin wall 8 cap strips Formats A amp D or one 1 optical adhesive film Formats C E F amp G per array Each 384 Well PCR Array format also includes one set of four 384 Well Plate EasyLoad Covers for each PCR Array provided in the package Technical Support 888 503 3187 US 301 682 9200 7 Methyl Profiler DNA Methylation PCR Array System NOTE The PCR Arrays can only be used in 96 well and 384 well real time PCR instruments The PCR Arrays cannot be used in the Cepheid SmartCycler the Roche L
13. Nase free H2O 470 ul i 590 ul 590 ul 1180 pl B 3 10 Mix tubes well by vortexing and briefly spin down the solution to the bottom of the tube B 3 11 Carefully add each cocktail to the appropriate wells of the 384 well plate using the provided 384 Well Plate Easy Load Covers as follows Place Cover 1 on the plate Add 10 uL of Mo cocktail to the open wells Odd number wells of rows A C E G I K M and O Remove amp discard the cover Place Cover 2 on the plate Add 10 uL of Ms cocktail to the open wells Even number wells of rows A C E G I K M and O Remove amp discard the cover Technical Support 888 503 3187 US 301 682 9200 19 Methyl Profiler DNA Methylation PCR Array System L DOZ Zr xe I NNM O0 gt Place Cover 3 on the plate Add 10 uL of Md cocktail to the open wells Odd number wells of rows B D F H J L N and P Remove amp discard the cover Place Cover 4 on the plate Add 10 uL of Msd cocktail to the open wells Even number wells of rows B D F H J L N and P Remove amp discard the cover 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 ZP NSE Ee EEEn ESE EEEE FEE A T T T T T T E E T B 3 12 After loading the plate carefully seal the plate or cap the wells Centrifuge the plate briefly to remove air bubbles at 1000 rpm for 1 minute Running the PCR Reactions B 3 13 To run the reactions use the two step program shown below for
14. a 24 genes 96 well PCR Array and one DNA sample Restriction digestion B 1 1 Perform the restriction digestions with the Methyl Profiler Enzyme Kit Catalog MeA 03 B 1 2 Prepare a reaction cocktail without enzymes as indicated below For this protocol it is recommended to use 1 ug genomic DNA The 5X digestion buffer should be thawed and vortexed well before use If any precipitates are present in the buffer make sure to continue mixing the buffer until precipitates dissolve RNase DNase free H20 5X Digestion Buffer 26 ul Genomic DNA 1 ug Final cocktail volume 120 ul B 1 3 After adding H2O to make the final cocktail volume 120 ul vortex to thoroughly mix the components and spin down briefly B 1 4 Set up four digestion reactions Mo Ms Md and Msd according to the following table AIl four tubes must contain equal amounts of genomic DNA Technical Support support sabiosciences com www sabiosciences com 12 Version 2 1 wo ms ma msa Enzyme A 1 pl 1 ul B 1 5 Pipette up and down to thoroughly yet gently mix the components Spin tubes briefly in microcentrifuge B 1 6 Incubate all four tubes at 37 C for 6 hours in a heating block or thermal cycler The reaction can also be performed overnight B 1 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 minutes B 1 8 The reactions are now ready for use or storage at 20 C If samples are stored pl
15. all cyclers Note the unique cycling conditions necessary to ensure proper performance of the assay 40 Haina cuve seimeni According to instrument g g recommendations The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle B 3 14 After the run the data can be analyzed as described in section V Technical Support support sabiosciences com www sabiosciences com 20 Version 2 1 B 4 Using a 24 genes 384 well PCR Array and four DNA samples Restriction digestion B 4 1 Perform the restriction digestions with the Methyl Profiler Enzyme Kit Catalog MeA 03 B 4 2 Prepare a reaction cocktail without enzymes as indicated below For this protocol it is recommended to use 0 5 pg genomic DNA per sample The 5X digestion buffer should be thawed and vortexed well before use If any precipitates are present in the buffer make sure to continue mixing the buffer until precipitates dissolves RNase DNase free H20 5X Digestion Buffer p27 ul Genomic DNA 0 5 ug H Final cocktail volume B 4 3 After adding HzO to make the final cocktail volume 125 ul vortex to thoroughly mix the components and spin down all four tubes briefly B 4 4 Set up four digestion reactions Mo Ms Md and Msd for each DNA sample according to the following table If all done at once you will have 4 digestion tube
16. cutive Way Frederick MD 21703 USA CONTENTS Background and Introduction 4 ll Materials Provided 7 lll Additional Materials Required 8 IV Protocols 10 A Tips for a Successful Assay 10 B Assay setup protocols 11 1 Signature 24 Gene Panel 96 well Array One Sample 12 2 Complete 96 Gene Panel 96 well Array One Sample 15 3 Complete 96 Gene Panel 384 well Array One Sample 18 4 Signature 24 Gene Panel 384 well Array 4 Samples 21 V Data analysis 25 VI Troubleshooting and Frequently Asked Questions 29 RT and Methyl Profiler are trademarks of SABiosciences Corporation ABI is a registered trademark of Applied Biosystems BioRad and iCycler are registered trademarks of BioRad Laboratories Inc MyiQ IQ Chomo and Opticon are trademarks of BioRad Laboratories Inc LightCycler is a registered trademark of Roche Applied Sciences SYBR is a registered trademark of Molecular Probes mastercycler is a registered trademark of Eppendorf SmartCycler is a registered trademark of Cepheid Rotor Gene is a trademark of Corbett Research LIMITED PRODUCT WARRANTY This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABioscience Corporation sha
17. d caused incomplete digestion e Incorrect Incubation Be sure to use at least a six hour incubation time at 37 C Conditions as well as the tubes with recommended size in the protocol Switch to an overnight incubation if a shorter time was previously used and caused incomplete digestion Technical Support 888 503 3187 US 301 682 9200 29 Methyl Profiler DNA Methylation PCR Array System Comments and suggestions 2 High Mock Digestion M C Values from Most if Not All Genes a Not Enough DNA Used in Be sure to use at least the amount of DNA recommended the Digestion in this protocol and to use the recommended methods and instruments to determine DNA concentrations Also be sure to include any RNase treatment steps recommended in the procedure of your chosen DNA isolation kit b Degraded DNA DNA samples are contaminated by microbes due to improper storage of your DNA samples such as at 4 C Always store your DNA samples at 20 C more than 2 years and at 80 C indefinitely c Improper PCR Array or Storing these components at inappropriate temperature Master Mix Storage for extended period reduces their activity and PCR amplification efficiency d Incorrect Real Time PCR Be sure that you used the correct cycling program that Cycling Program Used includes 10 minutes at 95 C which is required to fully activate the Hot Start enzyme in the RT SYBR Green qPCR Master Mix 3 All Four Digests M Ms Mg
18. e the C values from your instrument software to a blank Excel spreadsheet following the instructions provided by your instrument manufacturer 3 Excel Based Data Analysis Template a Download the Methyl Profiler PCR Array Excel based data analysis template that matches the gene panel and plate format that you used from our website at http www sabiosciences com methylationdataanalysis php Or click the DNA Methylation Data Analysis link found in the lower Product Support section of the gray right hand sidebar of any DNA Methylation web page Paste in your C value data and analyze the automatically generated results by simply following the directions in the Instructions worksheet of the Excel file 3 Data Quality Control a Mock Digest M C Values The C values of the mock digests for all genes should be within a range of 21 to 29 cycles if the recommended amounts of genomic DNA were used b Single Enzyme Digest Ms and Mg C Values Technical Support 888 503 3187 US 301 682 9200 25 Methyl Profiler DNA Methylation PCR Array System The C values of the Ms and Mg digests should be between the values of the mock and double digests depending on the methylation status of the DNA samples c Double Digest Msa Ci Values The C values of the double digests should be higher than the C values for the mock digest or single digests for the same gene d Enzyme Digestion Efficiency The difference in C
19. ease remember to vortex to thoroughly mix the samples after thawing Spin down briefly before proceeding to the next step Setting up the PCR Reactions B 1 9 Prepare individual PCR cocktails in a 1 5 mL tube for each of the four reactions Mo Ms Md and Msd from the previous step following the table below Technical Support 888 503 3187 US 301 682 9200 13 Methyl Profiler DNA Methylation PCR Array System io me wa Mea B 1 10 Mix tubes well by vortexing and briefly spin down the solution to the bottom of the tube B 1 11 Carefully add 25 ul of the Mo cocktail to each well in rows A amp B of the 96 well PCR Array 25 ul of the Ms cocktail to each well in rows C amp D 25 ul of the Md cocktail to each well in rows E amp F and 25 ul of the Msd cocktail to each well in rows G amp H as indicated below g 5S wel 1 2 3 4 5 6 7 8 9 10 11 12 a B 14 G15 G16 G17 G18 G19 G20 G21 22 G23 G24 u M Meg i B 1 12 After loading the plate carefully seal the plate or cap the wells Centrifuge the plates briefly to remove air bubbles at 1000 rpm for 1 minute Running the PCR Reactions B 1 13 To run the reactions use the two step program shown below for all cyclers Note the unique cycling conditions necessary to ensure proper performance of the assay Technical Support support sabiosciences com www sabiosciences com 14 Version 2 1 97 C 15 seconds 40
20. ell plate using the provided 384 Well Plate Easy Load Covers as follows Place Cover 1 on the plate Add 10 uL Mo cocktail to the open odd numbered wells of rows A amp C for Sample 1 E amp G for Sample 2 amp K for Sample 3 and M amp O for Sample 4 Remove amp discard the cover Technical Support support sabiosciences com www sabiosciences com 22 Version 2 1 Place Cover 2 on the plate Add 10 uL Ms cocktail to the open even numbered wells of rows A amp C for Sample 1 E amp G for Sample 2 amp K for Sample 3 and M amp O for Sample 4 Remove amp discard the cover Place Cover 8 on the plate Add 10 uL Md cocktail to the open odd numbered wells of rows B amp D for Sample 1 F amp H for Sample 2 J amp L for Sample 3 and N amp P for Sample 4 Remove amp discard the cover Place Cover 4 on the plate Add 10 uL Msd cocktail to the open even numbered wells of rows B amp D for Sample 1 F amp H for Sample 2 J amp L for Sample 3 and N amp P for Sample 4 Remove amp discard the cover Well 1 2 3 a 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 B 4 12 After loading the plate carefully seal the plate or cap the wells Centrifuge the plate briefly to remove air bubbles at 1000 rpm for 1 minute Running the PCR Reactions B 4 13 To run the reactions use the two step program shown below for all cyclers Note the unique cycling conditions necessary to ensure proper performance
21. ences com www sabiosciences com 26 Version 2 1 representation of the data using our developed Hierarchical Clustering method http www sabiosciences com dna_ methylation data_analysis php b Significance of Methylation Results The level of HM methylation considered to be significant potential positive marker for hyper methylation must be defined by the researcher However the same principles used to define the significance of bisulfite based sequencing and real time PCR based methods apply to the Methyl Profiler PCR Arrays and qPCR Assays as well Alternatively to define whether your results are significant you might take the following into consideration Percent of Hyper methylated DNA In most cases only hyper methylated promoters will repress gene expression Therefore the minimum level of hyper methylation considered to be positive can be set at 10 to 20 similar as defined for bisulfite based PCR methods However this is dependent on the ratio target vs non target cells present in the sample i e normal cells mixed with cancerous cells The greater the extent of contamination the higher the threshold must be set Comparison between a Control and Experimental DNA Samples Such parallel analysis will allow you 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 Low Hyper meth
22. fits Fast reliable and quantitative No bisulfite conversion Ready to use Genomic DNA Enzyme Digestion gt Real Time PCR gt Straightforward data analysis Disease or Pathway focused Simultaneously detect methylation status of 24 96 genes Genome Wide Coverage Pre designed primers to detect the of methylation status of your gene of interest Technical Support 888 503 3187 US 301 682 9200 5 Methyl Profiler DNA Methylation PCR Array System How METHYL PROFILER WORKS 1 DNA Digestion Input genomic DNA is aliquoted into four equal 1 DNA Digestion portions Input DNA Digestion Buffer a Mock Digest M No enzymes are added in this reaction The product of the mock digestion represents the total amount of input DNA for real time PCR detection Methylation dependent b Methylation Sensitive Digest M 37 C lete ovemighy Cleavage with a methylation sensitive enzyme will digest unmethylated and partially J AT SvBR Green qPCR Master Mix methylated DNA The remaining hyper 2 Real Time PCR PCR primer mix methylated DNA will be detected by real time ORE PCR Sensitive ssssssscseo Dependent bsssoooooo c Methylation Dependent Digest Ma Double yeessssseesee Cleavage with a methylation dependent mec eeseeess enzyme will preferentially digest methylated J DNA The remaining unmethylated DNA will 3 Data Analysis be detected by real time PCR d Double Digest Msa Both enzymes
23. fy SABiosciences User Manual n C biomol info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D Methyl Profiler DNA Methylation PCR Array System Fast and Reliable Quantitative DNA Methylation Analysis without Bisulfite Conversion See Purchaser Notification for limited use license and warranty information page 3 Part 1038A Version 2 1 3 10 2010 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Methyl Profiler DNA Methylation PCR Array System Methyl Profiler DNA Methylation PCR Array System Fast and Reliable Quantitative DNA Methylation Analysis Without Bisulfite Conversion User Manual For Catalog Numbers Prefixed by MeA H M Ordering and Technical Service Contact Information e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www sabiosciences com e E MAIL order sabiosciences com To place an order support sabiosciences com For technical support You may place orders by fax e mail or from our website Each order should include the following information e Your contact information name phone email address e Product name catalog number and quantity e Purchase order number or credit card information Visa or MasterCard e Shipping address e Billing address For more information visit us at www sabiosciences com SABiosciences Corporation 6951 Exe
24. ightCycler 2 0 or the Corbett Research Rotor Gene and Rotor Gene 6000 Ill Additional Materials amp Equipment Required A DNA Isolation Kit See Recommendations in the Protocol Section B DNA Methylation Enzyme Kit MeA 03 The MeA 03 kit contains all necessary components for the 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 12 DNA samples C RT SYBR Green qPCR Master Mixes These Master Mixes are ESSENTIAL for a complete and successful experiment Be sure to select the correct formulation size and quantity for your real time PCR instrument Catalog Master Mix PA 012 RT SYBR Green ROX qPCR Master Mix Designed specifically for all ABI and Stratagene Instruments and Eppendorf mastercycler ep realplex instruments with a ROX filter set PA 011 RT SYBR Green Fluorescein qPCR Master Mix Designed specifically for BioRad iCylcer MyiQ and iQ 5 instruments PA 010 RT SYBR Green qPCR Master Mix Designed specifically for instruments that do not require a reference dye like BioRad CFX96 BioRad Opticon Opticon 2 amp Chromo 4 MJResearch Roche LightCycler 480 System and Eppendorf mastercycler ep realplex instruments without a ROX filter set Technical Support support sabiosciences com www sabiosciences com For PCR
25. intermediate Yes Hyper methylated gt 60 _ percent methylated DNA with this methylated and unmethylated O percent technology methylated amounts of DNA in a sample are each directly and reliably detected The total amount of input DNA minus the hyper methylated and unmethylated DNA fractions yields the amount of intermediate methylated DNA between 0 and 60 percent methylated Lower extents of hyper methylation have been more and more correlated with biological phenotypes when concurrent with larger extents of intermediate methylation levels 7 Will pipetting error affect the The passive reference dyes in the PCR master MethyI Profiler PCR Array results mixes such as ROX and Fluorescein are used by the real time PCR instruments to normalize variation from well to well Therefore these systems tolerate volume variations caused by pipetting error or evaporation Even a 20 pipetting error causes only 0 05 cycle differences in C values 8 How can I prevent the evaporation Carefully and completely seal the PCR Array of reaction volume from the wells with the optical thin wall 8 cap strips or the optical adhesive film before placing it into your thermal cycler Technical Support support sabiosciences com www sabiosciences com 32 Version 2 1 If you have additional questions or technical difficulties please check our website www sabiosciences com for a more complete listing of Frequently Asked Questions FAQs or ca
26. ll not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER No rights are granted to use the components of this product for reproduction of any primer to modify product components for resale or to use this product to manufacture commercial products without written approval of SABioscience Corporation U S patents may cover certain isolated DNA sequences included in this product NOTICE TO PURCHASER Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 5 677 152 Claims 1 to 23 only 5 773 258 Claims 1 and 6 5 407 800 5 322 770 5 310 652 5 994 056 6 171 885 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount or product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims in US Patent No 6 814 934 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche
27. ll our Technical Support Representatives at 1 888 503 3187 or 301 682 9200 Technical Support 888 503 3187 US 301 682 9200 33 Methyl Profiler DNA Methylation PCR Array System NOTES Technical Support support sabiosciences com www sabiosciences com 34 Version 2 1 NOTES Technical Support 888 503 3187 US 301 682 9200 35 Methyl Profiler DNA Methylation PCR Array User Manual Part 1038A Version 2 1 3 10 2010 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O 22769 Hamburg b info biomol de iomo www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 _ www SABiosciences com support SABiosciences com
28. ns that the double digest contains only 100 x 2 C 100x2 5 3 125 percent of the DNA of the mock digest meaning that the reaction is therefore 100 3 125 96 875 percent complete 2 Can this technology reliably Thanks to the sensitive and quantitative nature of characterize heterogenous tissue real time PCR hyper methylated DNA can be samples detected even when the relevant cells are present at only five percent of a total population This level of sensitivity equals bisulfite sequencing and bisulfite PCR methods In contrast Methylation Specific PCR MSP only detects sequences when bisulfite conversion is 100 percent successful thereby having decreasing the assay sensitivity 3 How are the primers in the DNA The primers are designed around CpG islands methylation PCR Array designed known to be hyper methylated under relevant biological conditions or in relevant biological samples Besides the usual specific requirements that real time PCR primers must meet the amplicon sequence must contain restriction sites for both the methylation sensitive and methylation dependent enzymes As a result the amplicon lengths are around 150 to 400 bp but do not affect the PCR efficiency The design algorithm also accounts for the GC rich nature of genomic DNA sequences that tend to make primer design more difficult especially in and around CpG islands Each primer pair is also experimentally validated for a single dissociation curve
29. nts and lab ware Physically separate your workspace for PCR setup and post PCR work Before setting up an experiment decontaminate your PCR workspace and lab ware pipette barrels tube racks etc with 10 bleach and UV light Preferentially set up your reaction within a PCR workstation Do not peel the protective film from the PCR Array until immediately before use Close all tubes containing PCR products as soon as you finish transferring solutions Treat any lab ware tips or tubes containing PCR products or other DNA with 10 bleach before discarding 2 Genomic DNA Preparation and Quality Control High quality DNA is ESSENTIAL for obtaining accurate results The most important prerequisite for a successful DNA Methylation qPCR assay analysis is consistent high quality genomic DNA from every experimental sample Therefore sample handling and genomic DNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the DNA or decrease the restriction enzyme activities necessary for optimal DNA digestion Recommended Genomic DNA Preparation Method The QIAGEN Blood amp Tissue Kit is highly recommended for the preparation of genomic DNA samples Ensure that samples have been treated for the removal of RNA as RNA contamination will cause inaccuracies in DNA concentration measurements and can possibly affect restriction digestion efficiency DO NOT omit
30. of hyper methylated target DNA copies Cum is defined as Cum 2 S Cr 20M 1 Cp 24 MSM _ Ch 1 Cp in which Ct Ms represent the Ct values for the MSRE digest The amount of un methylated DNA Cum can be determined as 2 Cp 20M 1 Cp 27408 MaMo _ Ch 1 Cpr in which Ct Md represent the Ct values for the MDRE digest If the sum of Cum and Cum is smaller than 100 the amount of intermediately methylated DNA Cim equals 1 Cum Chm The Ct values can vary up to 1 0 cycle dependent on technical errors and well to well variations within Real Time PCR instruments In order to eliminated some of these variations it is recommended to use larger ACt numbers for the calculations of the relative amount of methylated and hyper methylated target DNA copies In our excel template when the ACt values are smaller than 1 0 the formulas are modified as indicated below ACt Ms Mo lt 1 0 and ACt Md Mo 2 1 0 then Crm 1 Cum ACt Md Mo lt 1 0 and ACt Ms Mo 2 1 0 then Cum 1 Chm ACt Md Mo lt 1 0 and ACt Ms Mo lt 1 0 then Cum Cum 50 methylation Technical Support support sabiosciences com www sabiosciences com 28 Version 2 1 VI Troubleshooting and FAQs A Troubleshooting Guide Comments and suggestions 1 Incomplete Restriction Enzyme Digestion C Msa C M lt 2 Incomplete digestion dramatically reduces the sensitivity of the Methyl Profiler PCR A
31. of the assay 97 C 40 72 C According to instrument Melting curve segment EEan E iDnE Technical Support 888 503 3187 US 301 682 9200 23 Methyl Profiler DNA Methylation PCR Array System The 10 minutes step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle B 4 14 After the run the data can be analyzed as described in section V Technical Support support sabiosciences com www sabiosciences com 24 Version 2 1 V Data analysis 1 Obtaining the raw Threshold Cycle C Values 2 After the cycling program is completed obtain the C values following the instructions provided by the manufacturer of your Real Time PCR Instrument We recommend manually setting the Baseline and Threshold Values according to the following directions Note that if you would like to compare multiple plates make sure that the settings for all plates are identical a 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 b 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 C Values Export and or copy and past
32. pin tubes briefly in microcentrifuge 1142y 112 ul B 2 6 Incubate all four tubes at 37 C for 6 hours in a heating block or thermal cycler The reaction can also be performed overnight B 2 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 minutes B 2 8 The reactions are now ready for use or storage at 20 C If samples are stored please remember to thoroughly mix the samples after thawing Spin down briefly before proceeding to the next step Setting up the PCR Reactions B 2 9 Prepare individual PCR cocktails in a sterile 14 mL Falcon tube for each of the four reactions Mo Ms Md and Msd from the previous step following the table below Mo Msd digest Total volume 2560 ul 2560 ul 2560 ul B 2 10 Mix tubes well by vortexing and briefly spin down the solution to the bottom of the tube 1160 pl 1160 ul 1280 ul 1280 ul 1160 ul 1280 ul 120 ul 2560 ul Technical Support support sabiosciences com www sabiosciences com 16 Version 2 1 B 2 11 Carefully add 25 ul of the Mo cocktail to each well in rows A amp B of the four 96 well PCR Arrays 25 ul of the Ms cocktail to each well in rows C amp D 25 ul of the Md cocktail to each well in rows E amp F and 25 ul of the Msd cocktail to each well in rows G amp H as indicated below Well 1 2 3 4 5 6 7 8 9 10 11 12 Mm B ms u B 2 12
33. r make sure to continue mixing the buffer until precipitates dissolves RNase DNase free H20 5X Digestion Buffer 100 ul Genomic DNA 2 ug Final cocktail volume 470 ul B 3 3 After adding H2O to make the final cocktail volume 470 ul vortex to thoroughly mix the components and spin down briefly B 3 4 Set up four digestion reactions Mo Ms Md and Msd according to the following table All four tubes must contain equal amounts of genomic DNA RNase DNase free H2O Cocktail from previous step 116 ul 116 ul 116 ul Enzyme A 2 ul Total volume 120 ul 120 ul 120 ul B 3 5 Pipette up and down to thoroughly yet gently mix the components Spin tubes briefly in microcentrifuge Technical Support support sabiosciences com www sabiosciences com 18 Version 2 1 B 3 6 Incubate all four tubes at 37 C for 6 hours in a heating block or thermal cycler The reaction can also be performed overnight B 3 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 minutes B 3 8 The reactions are now ready for use or storage at 20 C If samples are stored please remember to thoroughly mix the samples after thawing Spin down briefly before proceeding to the next step Setting up the PCR Reactions B 3 9 Prepare individual PCR cocktails in a 1 5 mL tube for each of the four reactions Mo Ms Md and Msd from the previous step following the table below 470 ul 470 ul RNase D
34. rray Complete digestion is guaranteed when using the Methyl Profiler DNA Methylation Enzyme Kit when the instructions are followed carefully The most common reason for incomplete digestion is poor DNA sample quality Common reasons for incomplete digestion are a Low Restriction Enzyme Be sure that the Methyl Profiler DNA Methylation Activity Enzyme Kit has not expired Be sure to use the correct amount of both enzymes recommended in this protocol for the DNA amount used b RNA Contamination in the RNA contamination inhibits restriction enzyme DNA DNA Samples digestion while also causing an overestimation of the DNA concentration Be sure to include any RNase treatment steps recommended in the procedure of your chosen DNA isolation kit c Other Contaminants in the DNA prepared from difficult organ tissues may contain DNA Samples 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 isolation d Too Much DNA Used in Be sure to use at least a four hour incubation time at the Digestion 37 C as well as the tubes with recommended size in the protocol Switch to an overnight incubation if a shorter time was previously used an
35. s for each DNA sample a total of 16 tubes All four digestion tubes must contain equal amounts of genomic DNA Mo Me ma Mea Enzyme A Technical Support 888 503 3187 US 301 682 9200 21 Methyl Profiler DNA Methylation PCR Array System B 4 5 Pipette up and down to thoroughly yet gently mix the components Spin tubes briefly in microcentrifuge B 4 6 Incubate all four tubes at 37 C for 6 hours in a heating block or thermal cycler The reaction can also be performed overnight B 4 7 After incubation stop the reactions by heat inactivating the enzymes at 65 C for 20 minutes B 4 8 The reactions are now ready for use or storage at 20 C If samples are stored please remember to thoroughly mix the samples after thawing Spin down briefly before proceeding to the next step Setting up the PCR Reactions B 4 9 Prepare individual PCR cocktails in a 1 5 mL tube for each of the four reactions Mo Ms Md and Msd from the previous step following the table below Alternatively the H2O and PCR Master Mix can be mixed together and then carefully aliquoted into the required amount of tubes for 4 samples this will be 16 tubes where after the digestion mixtures from the previous step can be added To me ma os B 4 10 Mix tubes well by vortexing and briefly spin down the solution to the bottom of the tube B 4 11 Carefully add each cocktail to the appropriate wells of the 384 w
36. the recommended RNase treatment step to remove RNA If genomic DNA samples need to be harvested from biological samples where kits are not available please contact Technical Support representatives for suggestions For best results resuspend or dilute all DNA samples in DNase free water or alternatively in DNase free 10 mM Tris buffer pH 8 0 without EDTA Technical Support support sabiosciences com www sabiosciences com 10 Version 2 1 e Measurement of DNA concentration and purity by UV spectrophotometry Prepare dilutions of your genomic DNA samples and measure absorbance in DNase free 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH Recommended rations and values A260 A230 gt 1 7 A260 A280 gt 1 8 A260 concentration gt 4ug mL e DNA concentrations needed 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 clinical tumor samples whereas heavy non tumor cell contamination is expected such as blood or stromal cells etc However maintain the specific enzyme to DNA ratios outlined below for each assay and purchase additional qPCR plates to ensure assay consistency 3 Methyl Profiler Enzyme Kit MEA 03 Handling Guide Important note e DONOT VORTEX ENZYMES Enzyme
37. ylation lt 10 and High Intermediate Methylation gt 60 Intermediate 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 one should consider measuring the corresponding expression levels and compare those with the expression levels in the appropriate controls 5 The AC Data Analysis Method The advantage of using Real Time PCR for the detection of remaining target DNA copies is the use of the cycle threshold C values which are inversely proportional to the amount of target DNA in the sample Also the product amount doubling after each cycle allows the total input Cmo to be defined as Cmo 9 CiMo A specific data analysis excel template was developed in order to calculate the relative amount of methylated DNA in each of the four assays using the ACt method The amount of remaining DNA in each digest is normalized to the total amount of input DNA mock digest Therefore the relative amount of target DNA copies that are resistant to enzyme digestion Cr are defined as Cp 2 C sd CtMo _ g ACt Msd Mo ih which Ct Msd represents the Ct values for the double digest assay This amount is subtracted as background signal in the calculations Technical Support 888 503 3187 US 301 682 9200 27 Methyl Profiler DNA Methylation PCR Array System The amount
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