TranSignalTM Promoter Methylation Array
Contents
1. 60 ml of 1X Blocking Buffer To 30 ml of deionized H O add 30 ml of 2X Blocking Buffer provided Mix well and store at 4 C 180 ml of 1X Wash Buffer To 135 ml of deionized H O add 45 ml of 4X Wash Buffer provided Mix well and store at room temperature RELATED PRODUCTS Please visit our website at www panomics com for the most up to date information about our products Products Size Catalog TranSignal Promoter Methylation Array Tkit TranSignal Promoter Methylation Array Refill 1 kit TranSignal Promoter Methylation Array 11 Refill 1 kit For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 16 TranSignal Promoter Methylation Arrays APPENDIX B Schematic diagram of the TranSignal Promoter Methylation Array survivin P27KIP1 STATS P2TKIP1 STATSa STAT MUC2 STAT POUSF1 Ras NPAT NPAT K x 5 E x 5 KIR2DLA MLCI CFTR KIRZOLA CDKN2A JUNG 2 E CAD 500 E CAD 500 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 17 APPENDIX D Stripping Procedure for TranSignal Arrays Note We do not encourage stripping the TranSignal array membranes more than two times Procedure 1 Wash membranes in 0 4M NaOH at 45 C for 30 min 2 W2. PREPARING GENOMIC DNA FROM CELLS OR TISSUES Genomic DNA can be prepared using a commercially available kit such as Panomics Genomic DNA Extraction Kit Cat AY2005 For best results your DNA should have a concentration of 100 500 ng ml 5 FRAGMENTATION OF GENOMIC DNA In this section you will digest the genomic DNA with Msel restriction enzyme to produce small fragments of DNA 200 bp that retain the CpG islands 5 1 Setup the following restriction digest Genomic DNA 200 ng ul 10 ul 10X NE Buffer 2 2 ul For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 8 TranSignal Promoter Methylation Arrays 5 2 5 3 54 5 5 5 6 5 7 5 8 6 Msel New England lul dH 0 7 ul Total Volume 20 ul Mix well by pipetting and incubate at 37 C for 2 hours Add 100 ul PB Buffer to the digest reaction and transfer 120 ul of this reaction mix to the DNA purification column Note Ensure that the yellow DNA purifiaction columns are used for this step Bind the DNA to the column centrifuge at 10 000g for 30 60 s Discard flow through Add 750 ul PE Buffer and centrifuge at 10 000g for 30 60 s Note Ensure that the correct wash buffer is used for this step Discard the flow through and centrifuge the column at maximum speed for 1 min Elute the DNA by adding 20 ul dH 0 to the center of the column membrane and let the column stand for 1 min Then centrifuge the
3. scription factors through comparison of two or more samples Follow these guidelines to analyze your results 11 1 Acquire the images using either x ray film or a chemiluminescence imaging system For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 11 2 11 3 11 4 TranSignal Promoter Methylation Arrays Adjust the exposure time such that the majority of the spots have equal signal intensity 11 2 1 If you are acquiring your image with an imaging system such as FlourChem measure the density of the spots and convert denisty to numbers using applicable software Purchase of a FlourChem Imaging System includes software that allows the denisty of each spot to be measured At Panomics we use the local area surrounding the individual spots for background subtraction 11 2 2 If you are using x ray film obtain an electronic imgage of your blot Then analyze the denisty of each spot using software with this ability Save data in an Excel spreadsheet and calculate the ratio of the data collected from the images For example if your experiment contains one control and two experimentals you may want to collect the ratios of sample 1 vs sample 2 and sample 1 vs sample 3 and perhaps sample 2 vs sample 3 Any spots with two fold increase or decrease are considered significant and should be confirmed by EMSA and or luciferase reporter assay NOTE During the quantification
4. 14 APPENDIX B Schematic diagram of the TranSignal Promoter Methylation Array l 15 APPENDIX C Schematic diagram of the TranSignal Promoter Methylation Array II 16 APPENDIX D Stripping procedure for TranSignal 17 APPENDIX E TranSignal Protein DNA Array 18 Trademarks Patents and Limited Warranty Panomics and TranSignal are trademarks of Panomics Inc FluorChem is a registered trademark of Alpha Innotech Corporations Hyperfilm is a trademark of Amersham Pharmacia Corporation This product is intended for research purposes only Panomics products may not be resold modified for resale or used to manufacture commercial products without written approval by Panomics Inc Panomics Inc warrants that the performance of this kit meets Panomics performance specifications from the time of shipment until the expiration date if stored under the recommended conditions Panomics disclaims all other warranties either express or implied including without limitation and implied merchantability or fitness for a particular purpose Under no circumstances shall Panomics be liable for any damages arising out of the use of the materials 2001 2005 C Panomics Inc All rights reserved PD UM012505 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Me
5. process some spots may show a two fold increase or decrease without a visible spot present on your membrane These data points are insignificant and should be considered background on the arrays For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 13 14 TranSignal Promoter Methylation Arrays 12 TROUBLESHOOTING GUIDE Problem Cause Recommendation Uneven background Substrate is not evenly Do not use thin cling wrap distributed on the membrane materials during the overlay procedure Re detect with substrate that is evenly covering the membrane surface High Background Incubation with substrate is Expose longer 10 to 15 min too long Incubation should not exceed 5 min Signal is too weak The yield of recovered DNA using control nuclear probe is low extract provided Check the concentration of your nuclear extract For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 15 APPENDIX A Recipes amp instructions for diluting stock solutions SECTION 6 300 ml of 2X SSC 0 5 SDS Hybridization Wash I To 262 5 ml of deionized H O add 30 ml of 20X SSC provided and 7 5 ml of 20 SDS provided Mix well 300 ml of 0 1X SSC 0 5 SDS Hybridization Wash II To 291ml of deionized H O add 1 5 ml of 20X SSC provided and 7 5 ml of 20 SDS provided Mix well SECTION 7
6. DNA is incubated with a methylation binding protein which forms a protein DNA complex These complexes are separated and methylated DNA is isolated 3 The methylated DNA is labeled with biotin dCTP via PCR and these probes are hybridized to the methylation array For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 5 CH3 CH3 CH3 Restriction digest eT CH3 RSA MBP Spin column separation Isolated adaptor c x p p Hybridize Signals correspond to specific methylated promoter Figure 1 Schematic flow chart of the TranSignal Promoter Methylation Array procedure For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 6 TranSignal Promoter Methylation Arrays 2 MATERIALS PROVIDED Box 1 Array Membranes amp Hybridization Reagents 4 TranSignal Promoter Methylation Array 3 each DNA Purification Columns 6 Columns DNA Purification Column Collections Tubes 6 Tubes Separation Column 3 each 1X Column Incubation Buffer 2 ml 1X Column Wash Buffer 10 ml 1X Column Elution Buffer 200 pl Hybridization Buffer 15 ml 20 SDS 15 ml Streptavidin HRP conjugate 60 ul Solution I 600 ul Solution II 600 pl Solution III 5 ml PE Buffer 5 ml PB Buffer 650 ul Box 2 Reaction Kit 20 Di
7. TranSignal Promoter Methylation Array Cat MA7010 Product User Manual Released 05 25 05 SS EIS Pa nom ics Transcending boundaries Panomics Inc 2003 East Bayshore Road Redwood City 94063 USA Tel 650 216 9736 or 877 726 6642 PANOMIC Fax 650 216 9790 www panomics com 2 TranSignal Promoter Methylation Arrays Contents 1 1 00 110 2 3 2 MATERIALS PROVIDED 6 3 ADDITONAL MATERIALS 0 6 4 PREPARING GENOMIC DNA FROM CELLS OR TISSUES 7 5 FRAGMENTATION OF GENOMIC 7 6 LIGATION OF PCR ADAPTORS TO DNA FRAGMENTS 8 7 ISOLATION OF METHYLATED DNA 8 8 BIOTINYLATION OF METHYLATED DNA FRAGMENTS 10 9 HYBRIDIZATION RU Ed PORE RUPEE A DE 10 rc 11 TA RESULTS amp ANALYSIS siste RR ETE REOR HERO URS UR RU n 12 12 TROUBLESHOOTING 6 0 13 APPENDIX A Recipes amp instructions for diluting stock solutions
8. ash membranes in 0 2M Tris HCL pH 7 6 0 1X SSC and 0 1 SDS at 45 C for 15 min 3 To ensure that stripping was successful run it through the standard chemiluminescence detection procedure as described in this user manual 4 After detection wash the membrane in 1X Washing Buffer at 42 C for 30 min 5 Membranes are ready for hybridization or dry the membrane in an 80 C incubator for later use For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 18 TranSignal Promoter Methylation Arrays APPENDIX E TranSignal Protein DNA Array Grid Photocopy this page onto separate piece of paper or transparency film Note that the notch is at the top right hand corner 1234567 8 9101112134 15 16 17 18 19 20 21 22 23 4 1 v2 c3uuLL c57T 2 2Zzzco a For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 19 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com
9. column at maximum speed for 1 min LIGATION OF PCR ADAPTORS TO DNA FRAGMENTS In this section you will add the adaptors for future PCR steps to the restricted ends of the DNA fragments 6 1 6 2 6 3 6 4 Add the following components to a 0 5 ml microfuge tube Digested DNA from step 5 8 10 ul Linker 2 ul 10X ligase binding buffer 2ul dH 0 4ul Total Volume 18 ul Heat the samples at 50 C for 3 min Lower the temperature to 25 C Add 2 ul of ligase For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 9 6 5 Mix components by pipetting and incubate at room temperature for 30 min 6 6 Repeat steps 5 3 5 8 7 ISOLATION OF METHYLATED DNA FRAGMENTS In this section you will isolate the methylated DNA fragments from the non methylated fragments All centrifuge steps should be carried out on a regular benchtop centrifuge at 7 000 rpm at 4 C unless otherwise stated 71 Add the following components to 0 5 ml microfuge tube MBP 2 ul Linker DNA from step 6 8 14 ul 5X Binding Buffer 4 ul Total Volume 20 ul 7 2 Mix components by pipetting and incubate at 15 C for 30 min 7 3 Meanwhile wash the Protein Spin Column by adding 500 chilled 1X Column Incubation Buffer and centrifuging at 7 000 rpm for 30 sec at 4 C Note Ensure the pink Protein purification columns are used for this step 74 Add 20ul 1X Column Incuba
10. eserve methylation of the genomic DNA The first conventional method employed for the analysis of DNA methylation used methylation sensitive restriction enzymes coupled with Southern blot analysis of DNA fragments This approach is reliable but time consuming and requires a substantial amount of high quality genomic DNA Recently another method has been adopted to study promoter methylation This uses sodium bisulphate conversion of methylated bases in conjunction with PCR amplification and sequencing of the PCR product This method is very tedious and inconsistent and all of the conventional methods are time consuming allowing the analysis of one promoter at a time Panomics has developed a new technology for the high throughput analysis of promoter methylation which simultaneously profiles the methylation status of 100 different promoter regions from one sample For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 3 4 TranSignal Promoter Methylation Arrays Principle of TranSignal Technology TranSignal Arrays use a proprietary patent pending technology developed by Panomics for the high throughput analysis of promoter methylation The TranSignal procedure is simple and straightforward Figure 1 Three basic steps are involved 1 Genomic DNA is digested with a restriction enzyme to isolate DNA with CpG islands The digests are purified and adapted with linkers 2 The adapted
11. o a clean microcentrifuge tube Dilute 20 ul of the stock Streptavidin HRP conjugate with 1 ml of the 1X Blocking Buffer Vortex the diluted streptavidin and transfer back into the 1X Blocking Buffer from Step 8 2 containing the membrane Add the diluted conjugate to the container making sure not to pour directly on the membrane Continue shaking the membrane for 15 min at room temperature Decant the diluted Streptavidin HRP solution Wash each membrane three times at room temperature with 20 ml of 1X Wash Buffer each for 8 min Remove excess substrate by gently applying pressure over the top sheet Using a paper towel remove excess substrate that might be remaining on the surface of the sheets Expose the membranes using either Hyperfilm ECL 2 10 min or a chemiluminescence imaging system 5 15 min such as the FluorChem imager from Alpha Innotech Corp In either case we recommend that you try several different exposure times Obtain quantitative analysis if desired If you are using a chemiluminescence imaging system follow the instructions that are provided with that system s software If you are using Hyperfilm ECL you will need to scan the film to obtain numerical data for comparison RESULTS amp ANALYSIS The main advantage of the TranSignal Protein DNA Arrays is that you can simultaneously analyze multiple transcription factors TranSignal Arrays give you quick answers when you want to identify those activated tran
12. prepared in Section 8 2 by heating it at 96 C for 5 min and quickly chill in ice for 2 min Add half of the denatured probe to each hybridization bottle and hybridize at 42 C overnight Decant the hybridization mixture from each hybridization bottle and wash each membrane as follows See Appendix A for recipes 9 4 1 Add 50 ml of prewarmed Hybridization Wash I incubate at 42 for 20 min in a rotating hybridization oven Decant liquid and repeat wash 9 4 2 Add 50 ml of prewarmed Hybridization Wash II incubate at 42 C for 20 min in a rotating hybridization oven Decant liquid and repeat wash DETECTION IMPORTANT Do not allow the membrane to dry during the detection 10 1 Using forceps carefully remove each membrane from its hybridization bottle and transfer to a new container containing 20 ml of 1X Blocking Buffer each membrane needs its own container We use a container that is equivalent to the size of a 200 ul pipet tip For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 12 TranSignal Promoter Methylation Arrays 10 2 10 3 10 4 10 7 10 8 container 4 5 x 3 5 Block the membrane by incubating at room temperature with the 1X Blocking Buffer for 15 min with gentle shaking Dilute 20 ul of Streptavidin HRP conjugate 1 1000 with the 1X Blocking Buffer from the blot container Prepare the dilution by removing 1 ml of the 1X blocking buffer t
13. stilled H O RNase DNase free 500 ul 2X Blocking Buffer 30 ml 1X Detection Buffer 60 ml 10X ligase Binding Bufer 10 ul Ligase 6 ul 5X Binding Bufer 25 ul MBP 6 ul Linker 15 ul Biotin dCTP labeling Mix 15 ul 2X PCR Buffer 80 ul 20X SSC 32 ml 4X Wash Buffer 45 ml 3 ADDITIONAL MATERIALS REQUIRED 3 1 Reagents and Solutions Genomic DNA Extraction kit e g Panomics Genomic DNA Extraction Kit Cat AY2005 Msel Restriction Enzyme Hybridization Wash I 2X SSC 0 5 SDS Hybridization Wash II 0 1X SSC 0 5 SDS For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal Promoter Methylation Arrays 7 e Deionized See Appendix A for recipes 3 2 Materials and Equipment 0 5 ml and 1 5 ml microfuge tubes Pipetman and tips Microcentrifuge Hybridization oven and bottles Stratagene Cat 401030 Note skirted centrifuge tubes with screw caps may be used in place of the hybridization bottles VWR Cat 21008 480 Hybridization bottle dimensions 150 mm x 35 mm diameter tubes e Plastic containers 4 5 x 3 5 equivalent to the size of a container for 200 ul pipet tips Shaker Plastic wrap plastic sheet protectors or overhead transparency used for detection see Section 7 6 Hyperfilm ECL Amersham Cat RPN3114K OR e Chemiluminescence imaging system e g FluorChem from Alpha Innotech Corp 4
14. thylation Arrays 1 INTRODUCTION DNA methylation is a commonly occurring modification of human DNA This modification involves the addition of a methyl group to cytosine residues at CpG dinucleotides a reaction that is catalyzed by DNA methyltransferase DNMT enzymes CpG dinucleotides are gathered in clusters called CpG islands which are unequally distributed across the human genome There are approximately 30 000 CpG islands in the genome and 50 6076 of these are within the promoter region of genes CpG islands are primarily unmethylated in normal tissues and the aberrant methylation of CpG islands is clearly related with diseases such as cancer Research shows that MeCP2 is a member of a family of proteins that selectively recognizes methylated CpGs The binding of these proteins to DNA leads to an altered chromatin structure which subsequently prevents the binding of transcription machinery and thus precludes gene expression The abnormal methylation causes transcriptional repression of numerous genes leading to tumor growth and development Studies of DNA methylation in cancer have uncovered new potential targets for the diagnosis prognosis and ultimately the treatment of human cancer There has been a delay in the appreciation of methylation as an important epigenetic event in cancer progression This has been due to the difficulties associated with the analysis of DNA methylation as standard molecular biology techniques do not pr
15. tion Buffer to the MBP DNA from step 7 1 Transfer all of this mix into the center of the Protein Spin Column 7 5 Incubate the Spin Column on ice for 30 min 7 6 Centrifuge column at 7 000rpm for 30 sec at 4 C and discard the flow through 7 7 Place the Spin Column in a new Spin Column Collection Tube provided 7 8 Add 6001 1X Column Wash Buffer to the Spin Column and incubate for 10 min on ice 7 9 Centrifuge column at 7 000rpm for 30 sec at 4 C and discard the flow through 7 10 Wash the column by adding 600ul 1X Column Wash Buffer to the Spin Column and centrifuging at 7 000rpm for 30 sec at 4 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 10 TranSignal Promoter Methylation Arrays 7 11 Repeat step 7 10 a further 3 times 7 12 Remove residual Wash Buffer by an additional centrifugation at 10 000rpm for 30 sec at 4 7 13 Add 20ul 1X Column Elution Buffer to the center of the Spin Column and incubate at room temperature for 5 min 7 14 Place the Spin Column in a clean 1 5ml microcentrifuge tube and centrifuge for 1 minute at 10 000rpm at room temperature 7 15 Place the microfuge tube containing the collected flow through on ice and use for further steps 8 BIOTINYLATION OF METHYLATED DNA FRAGMENTS In this step the purified methylated DNA fragments will be converted into biotinylated probes 8 1 the following components in a 0 5 ml microfuge
16. tube Methylated DNA from step 7 15 lul PCR Primers 3 ul biotin dCTP 5 ul 2X PCR buffer 25 ul dH 0 16 ul Total Volume 50 ul 8 2 Mix well by pipetting and carry out the following PCR steps for 30 cycles 30 cycles of the following steps 94 C 1 min 55 C 1 min 72 2 min 4 C Forever For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 9 TranSignal Promoter Methylation Arrays HYBRIDIZATION In this Section you will hybridize the labeled probe prepared in Section 8 to the array membrane Before you begin warm the hybridization buffer to 42 C in a water bath If you notice a cloudy or soapy appearance make sure the particulates are completely dissolved before proceeding with hy bridization It may require overnight heating in a water bath 9 1 9 2 9 3 9 4 Place each array membrane into a hybridization bottle Wet the membrane by filling the bottle with deionized H O Then carefully decant the water Be sure to place the membrane in the hybridization bottle such that the spotted oligos face the center of the tube away from the walls NOTE When the membrane is properly oriented the notched corner will be in the top right To each hybridization bottle that contains an array membrane add 3 5 ml of prewarmed Hybridization Buffer provided Place each bottle in the hybridization oven at 42 C for 2 hr Denature the biotin labeled PCR product
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