User Manual - Biomol GmbH
Contents
1. Figure 4 Histone Modifications and Gene Activity Antibodies from ChampionChIP Antibody Kits against modified histones H3K4me3 H3K27me3 and H3K9me3 or control IgG were used for chromatin immunoprecipitation from HeLa cells following the instructions specified in the User Manual Each ChIP DNA sample was analyzed by Real Time PCR using primers specific for the transcriptional active genes GAPDH RPL30 and ALDOA transcriptional inactive genes MYOD1 SERPINA1 silenced repeats SAT2 Sata and an open reading frame ORF free region IGX1A The results are shown as the percentage of co precipitated DNA relative to the input DNA The antibodies against H3K4me3 H3K27me3 and H3K9me3 specifically enrich genomic DNA at actively transcribed euchromatin transcriptional inactive euchromatin or silent heterochromatic region respectively In contract the control IgG showed minimum background among all the regions analyzed demonstrating the efficiency and specificity of the ChIP Assay on genomic DNA enrichment 2 Primer Sets for ChIP DNA enrichment QC The primers sets listed below are designed for ChIP DNA associated with modified Histone antibodies such as H8ac H3acK9 H4ac H3K4me2 3 H3K9me1 2 3 H3K27me3 H4K20me2 3 H2K79me3 Additional control primer sets are available for the Human and Mouse genome at http www sabiosciences com chipgradeantibody php pus Tie HUMAN MOUSE Active Gene GAPDH 41 GPH10001C 01A GPMO050651 01A Inactive2. 2 ACt Normalized IPD X 10095 Calculate the 96 Input Enrichment Specific Enrichment by subtracting the 96 Input of the control IP Input con from the 96 Input of Antibody IP 96 Input Ab Input Enrichment Input Ab Input con Background adjust the Ab IP DNA Ct values by subtracting the input normalized Control IP DNA Ct values from input normalized IP Ct values first AAC AACt IP ACt normalized IP ACt normalized Con The Fold Enrichment of a sample is calculated by the linear conversion of the background adjusted first AACt Fold Enrichment 2 Ct lIPP The difference in fold enrichment for each gene across two ChIP PCR Arrays or groups can be determined by dividing the fold difference of group 1 by the fold difference of group 2 Fold Difference Fold Enrichment G2 Fold Enrichment G1 9 AACE IP S2 AACt IP 1 Where group 1 G1 is the control and group 2 G2 is the experimental group OPTIONAL f the fold change is greater than 1 then the result may be reported as a fold up regulation If the fold change is less than 1 then the negative inverse of the result may be reported as a fold down regulation The fold change ratios may also be reported as it is Technical Support 888 503 3187 US 301 682 9200 25 ChampionChIP PCR Arrays Reproducibility of ChIP PCR Array amp Positive PCR Control PPC Examine the Ct values of the PPC reaction Any impurities in your C
3. Each IP fraction requires the ChIP Ready Chromatin from one million cells Divide the ChIP Ready Chromatin accurately and evenly into the follow equal IP fractions Control Fraction IgG NIS required Ab Fraction at least one required Anti RNA Polymerase Il Antibody optional The Pol Il Antibody is used as a positive control for a successful ChIP assay in combination with the ChIP PCR primers for the GAPDH proximal promoter This fraction can also provide a measurement of the transcriptional activity when used with other proximal promoter ChIP PCR Assays 5 Reverse Cross Linking Follow the recommendations in your ChIP kit or follow your own protocol 6 DNA Purification Technical Support 888 503 3187 US 301 682 9200 15 ChampionChIP PCR Arrays Follow your previously used methodology or the recommendations in your ChIP kit Adjust the final volume of each ChIP DNA sample to 200 pL using the elution buffer provided or DNase free H20 The quality of the genomic DNA from ChIP DNA sample can be determined in the following section ChIP DNA Quality Control 1 Specificity and Efficiency of ChIP Enriched DNA The results of ChIP Array are dependent on the DNA quantity enriched by ChIP Assay and most importantly on the specific localization of these enriched DNA bound to transcription factors or associated with modified histones For example it is well defined that many modified histone marks have distinct distribution pat
4. C E F and G per array 4 Each 96x4 Format 384 Well ChIP PCR Array Formats E and G includes one set of four 384EZLoad Covers Catalog PA 384 for each ChIP PCR Array provided in the package Each 384EZLoad Cover is for Single Use ONLY Technical Support support SABiosciences com www SABiosciences com Version 1 1 Ill Additional Materials Required A ChampionChIP One Day Kit Catalog Number GA 101 The One Day Kit contains sufficient reagents to perform 12 Chromatin Immunoprecipitations and DNA Purifications The Kit yields high quality ChIP DNA suitable as template for Real Time PCR analysis See Figure 4 on page 17 and Figure 5 on page 18 B Champion Antibody Kit Catalog Number GAH XXXX More information regarding the ChampionChIP Antibody Kit can be found at http www sabiosciences com chipgradeantibody php OR other ChIP Grade antibody of choice can be used See Page 12 C SABiosciences RT SYBR Green qPCR Master Mixes MANDATORY for a Complete and Successful Experiment Be sure to select the correct master mix appropriate for the Real Time PCR instrument available at your laboratory and the correct master mix volume for your chosen PCR Array format 1 96 Well PCR Arrays RT SYBR Green ROX qPCR Master Mix PA 012 Designed for All ABI and Stratagene Instrumentation Eppendorf mastercycler ep realplex Instruments with ROX filter set Catalog Number Size PA 012 4 For 4 ChIP PCR Array plates PA 01
5. ChIP DNA Sample Preparation 12 B Performing Real Time PCR 17 C ChIP PCR Array Data Acquisition Real Time 21 D ChIP PCR Array Data Analysis 23 VI Troubleshooting and Frequently Asked Questions 27 Appendix AACt Data Analysis Method amp Bibliography 28 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use 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 shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of ChampionChIP PCR Array includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any primer pair mix to modify kit components for resale or to use ChampionChIP PCR Array to manufacture commercial products without written approval of SABioscience Corporation No other license expressed implied or by estoppels is granted U S patents may cover certain isolated DNA sequences included in the ChampionChIP
6. Gene MYOD1 1 GPH10002C 01A GPM038008 01A Heterochromatin SAT2 1 GPH10003C 4 01A Heterochromatin IAP 1 GPM00002C 01A 3 ChIP DNA Quantity Needed for Real Time PCR QC Analysis A typical ChIP Assay yields 100 to 200 uL of IP DNA The fold enrichment is independent of the volume of ChIP DNA used as a template in the Real Time PCR assay provided that the ChIP DNA samples are of high quality and free of PCR inhibitors Figure 5 For QC Technical Support 888 503 3187 US 301 682 9200 17 ChampionChIP PCR Arrays analysis we recommend starting with 1 100 2 uL of purified ChIP DNA from either IP or Input faction a 25 uL Real Time PCR Assay 96 Well format or 1 200 1 uL for a 10 uL Real Time PCR Assay 384 Well format 35 Input Pol II 33 Control IgG Ct Values Volume in ul Figure 5 ChampionChIP One Day Kit ChIP DNA Free of Inhibitors of Real Time PCR ChIP Assay was performed with either RNA Polymerase II or Control IgG using ChampionChIP One Day Kit and RNA Polymerase II antibody Kit Different volume 0 5 1 0 2 0 and 4 0 uL of Input DNA diamonds and IP DNA from either Pol Il IP circles or IgG IP triangles were added to a Real Time PCR assay using primers for the proximal promoter region of GAPDH The calibration curve is generated based on the threshold cycle values The data was fitted according to a linear regression model resulting in an R of 0 9898 indicating a straight line This i
7. LightCycler 480 System Catalog Number Size PA 010 8 For 4 384 well ChIP PCR Array plates D Real Time PCR instrumentation and other equipment 1 For recommendations on specific Real Time PCR instruments thermal cyclers with Fluorescent detection see the list of master mixes and plate formats above 2 Calibrated Multi Channel Pipette 3 DNase free PCR Grade water H2O pipette tips and tubes NOTE The ChIP PCR Arrays can only be used in 96 well and 384 well Real Time PCR Instruments PCR Arrays cannot be used in the Cepheid SmartCycler the Roche LightCycler 2 0 or the Corbett Research Rotorgene Technical Support support SABiosciences com www SABiosciences com 10 Version 1 1 IV Protocol Preparing a DNase free Workspace and avoiding DNA Contamination For first time users it is recommended to read through the entire protocol before starting an experiment For experienced users a quick guide is included ChIP enriched genomic DNA samples are usually limited in quantities and susceptible to degradations by DNases Maintaining a DNase free environment is essential for successful and reproducible results For accurate and reproducible PCR results it is very important to avoid contamination of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed profiles and or false positive signals The most common sources of DNA contamination are the products of pre
8. in gene inactivation or silencing Chromatin Immunoprecipitation ChIP is a unique technology for the analysis of the in vivo association of a protein of interest with specific genomic regions in native chromatin microenvironments Current approaches for the analysis of ChIP enriched DNA samples rely mainly on site by site detection of limited genomic regions using PCR or screening large scale genomic regions by array hybridization ChIP on Chip or direct sequencing ChIP Seq Our ChampionChIP PCR Array ChIP PCR Array is a 96 well or 384 well PCR plate containing SYBR Green optimized primers specially designed for application focused panels of genomic sites allowing for a specific and quantitative analysis of ChIP DNA samples The ChIP PCR Array combines the advantage of Real Time PCR dynamic performance with the ability to detect ChIP enriched genomic DNA on multiple sites simultaneously The global profiling feature of the ChIP PCR Array eliminates the low throughput of single site by site detection The product greatly facilitates the profiling of ChIP DNA enriched for function or disease relevant genomic loci The results will aid in the understanding of the coordinated and dynamic regulation of multiple genes during cell development differentiation tumor genesis and possible other human diseases To complete the ChIP PCR Array procedure Figure 1 all you need to do is prepare your experimental samples about one million cells per Ch
9. positive calls See page 13 ChIP Preparation No action is needed when the IGXA Ct value of the Input fraction is within the range of 24 to 30 2 Ctand 96 Input values of the ChIP Fractions Input Fraction a b Individual Input C value C Input is generally 30 Numbers are listed within the calculations worksheet The Average Ci input C Input Ave must be lt 30 Numbers are listed within the QC Report worksheet The difference of C Input Ave among each group at the QC Report worksheet should be within a reasonable range generally the difference of C Input Ave 1 585 equal to 3 fold difference of the Input Fraction Technical Support support SABiosciences com www SABiosciences com 26 Version 1 1 Control IP Fraction IgG NIS Mock a Individual Control C value C Con in the calculations worksheet should be C Con lt 32 or gt C Input 2 b The average C Con or C Con Ave in the QC Report worksheet should be within a reasonable range generally C Con lt 32 or C Input 2 c Most importantly the individual Input Con IgG NIS mock background level in calculations worksheet should be in general lt 0 1 l Specific IP Fractions Antibodies of choice The specific IP C value C Ab for each genomic site is variable and depends on the experimental samples and antibody quality Nevertheless the C Ab value indicates the enrichmen
10. throughput profiling capabilities with Real Time PCR performance Technical Support 888 503 3187 US 301 682 9200 5 ChampionChIP PCR Arrays A CHROMATIN IP ChampionChIP One Day Kit amp Ab Kit Starting Material Tissues Cells 4k Crosslink amp Fragmentation ChIP Ready Chromatin Dilute Pre clear Aliquot Equal Volume into the Following Chromatin Fractions 196 IP Fractions 1x10 Cells per IP Fraction J Immunoprecipitation with Abs Control Input Fraction Ab Fractions Control Fraction l Reverse Crosslinking amp Purification I IP Process ChIP DNA Samples B Real Time PCR by Adding ChIP DNA and PCR Master Mix to ChIP PCR Array Plates C ChIP PCR Array Data Acquisition Figure 1 Overview of the ChIP PCR Array procedure Technical Support support SABiosciences com www SABiosciences com 6 Version 1 1 A A Gl G2 G3 G4 G5 G6 G7 G8 G9 B G13 G14 G15 G16 G17 G18 G19 G20 G21 C G25 G26 G27 G28 G29 G30 G31 G32 G33 D G37 G38 G39 G40 G41 G42 G43 G44 G45 G46 E G59 F G61 G62 G63 G64 G65 G66 G67 G68 G69 G70 G71 H Cl C2 cS c4 GS C6 C7 c8 G9SIECTIOSBPPG 1 2 3 4 5 8 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 G1 G1 62 G2 63 G3 G4 G4 G5 G5 G6 G6 G7 G7 G8 G8 G9 G9 G10 G10 G11 G11 G12 G12 PEELE EEE EEE EE EEE
11. 2 12 For 12 ChIP PCR Array plates PA 012 24 For 24 ChIP PCR Array plates RT SYBR Green Fluorescein qPCR Master Mix PA 011 Designed for BioRad iCylcer MyiQ and iQ 5 Instrumentation Catalog Number Size PA 011 4 For 4 ChIP PCR Array plates PA 011 12 For 12 ChIP PCR Array plates PA 011 24 For 24 ChIP PCR Array plates RT SYBR Green qPCR Master Mix PA 010 Specifically designed for instrumentation that does not require a reference dye BioRad CFX96 BioRad Opticon Opticon 2 amp Chromo 4 MJ Research Roche LightCycler 480 System Eppendorf mastercycler ep realplex Instruments without ROX filter set Catalog Number Size PA 010 4 For 4 ChIP PCR Array plates PA 010 12 For 12 ChIP PCR Array plates PA 010 24 For 24 ChIP PCR Array plates Technical Support 888 503 3187 US 301 682 9200 9 ChampionChIP PCR Arrays 2 384 Well PCR Arrays 96 x 4 Format RT SYBR Green ROX qPCR Master Mix PA 012 Specifically designed for All ABI and Stratagene Instrumentation Catalog Number Size PA 012 8 For 4 384 well ChIP PCR Array plates RT SYBR Green Fluorescein qPCR Master Mix PA 011 Specifically designed for Instrumentation that requires the Fluorescein Reference Dye Catalog Number Size PA 011 8 For 4 384 well ChIP PCR Array plates RT SYBR Green qPCR Master Mix PA 010 Specifically designed for instrumentation that does not require a reference dye BioRad CFX384 Roche
12. C10 to ensure the specificity and efficiency of the enrichment Well H11 and well H12 contain replicate Positive PCR Controls PPC The replicate PPC control wells also indicate inter well and inter plate consistency B Catalog 384 Well ChIP PCR Array The 384 well format of the PCR Arrays includes four replicates of the same 96 well format in which each set of wells contains the same primer set represented by the 96 well designations For example a set of wells A1 A2 B1 B2 the same primer set labeled with G1 Wells A1 through N24 contain a Real Time ChIP PCR assay for genomic sites from the same biological pathway disease state or genes that are functionally related Wells O1 to O10 through P1 to P10 contain control Real Time ChIP PCR assays C1 C10 to ensure the specificity and efficiency of the enrichment Wells O11 O12 and P11 P12 contain replicate Positive PCR Controls PPC The replicate PPC control wells also indicate inter well and inter plate consistency Customized ChIP PCR Arrays will have a customer specified layout The product specification sheet enclosed with the arrays will reiterate the layout and will list the genes included on the array Technical Support 888 503 3187 US 301 682 9200 1 ChampionChIP PCR Arrays Il Materials Provided All components included in this kit are shipped at ambient temperature but must be stored at 20 C upon receipt Quality is guaranteed up to 6 months of receipt if sto
13. Data Analysis Template Excel or Web based Utilities See Section D Data analysis NOTE Save the PCR settings as an instrument file for next time performance when possible 4 Recommended Real Time PCR Quality Assessment a Amplification Curve Shape The Baseline Cycle Range for all reactions should have a constant flat low level of fluorescence in the Linear View of the amplification curve In the log scale view the slopes and therefore the amplification efficiencies of all assays should also be parallel to each other to be comparable b Dissociation Melting Curve Confirm that each reaction produces a single specific product as indicated by a single Dissociation Curve peak at a melting temperature Tm greater than 75 C Also confirm that the peak Tm values are within 1 0 C of each other for all reactions with the same ChIP PCR primer For instrument specific melt curve analysis settings please refer to the corresponding Instrument Setup Guide for your instrument at http sabiosciences com pcrarrayprotocolfiles php General Protocol for a melting curve Run a melting curve program immediately after the above cycling program and generate a first derivative dissociation curve for each well in the entire plate using your instrument s software No more than one peak should appear in each reaction at temperatures greater than 80 C If your instrument does not have a default melting curve program run the following program i
14. EEE EE G13 G13 G14 G14 G15 G15 G16 G16 G17 G17 G18 G18 G19 G19 G20 G20 G21 G21 G22 G22 G23 G23 G24 G24 Sr ene ee be ee eee Eee Ep eec ee ee G25 G25 G26 G26 G27 G27 G28 G28 G29 G29 G30 G30 G31 G31 G32 G32 633 G33 G34 G34 G35 G35 G36 G36 Bar IEEE MEE G37 G37 G38 G38 G39 G39 G40 G40 G41 G41 G42 G42 G43 G43 G44 G44 G45 G45 G46 G46 G47 G47 G48 G48 Ee eee eee Eee eque G49 G49 G50 G50 G51 G51 G52 652 G53 G53 G54 G54 G55 G55 G56 G56 G57 G57 G58 G58 G59 G59 G60 G60 Ec pee esee e G61 G61 G62 G62 G63 G63 G64 G64 G65 G65 G66 G66 G67 G67 G68 G68 G69 G69 G70 G70 G71 G71 G72 G72 pe pop es ee eee G73 G73 G74 G74 G75 G75 G76 G76 G77 G77 G78 678 G79 G73 G80 G80 G81 G81 G82 G82 G83 G83 G84 G84 ea REESE C1 C1 C2 C2 C3 C3 C4 j C4 C5 j C5 C6 C6 C7 C7 C8 C8 C9 10 C10 PPC PPC PPCJPPC Ozzrxc crgommocoocmz P Figure 2 Layout of the Cataloged ChIP PCR Arrays A Catalog 96 well ChIP PCR Arrays Wells A1 through G12 contain a Real Time ChIP PCR assay for genomic sites from the same biological pathway disease state or genes that are functionally related Wells H1 through H10 contain control Real Time ChlP PCR assays C1
15. H3ac requires fewer cells but no less than 0 5 million cells Lower amounts of starting material will yield a smaller number of positive calls and will increase false negative calls On the contrary lower abundance of the protein associated DNA or lower efficient antibodies will require more cells A high amount of starting material will yield a greater number of positive calls though it will also increase background levels and possibly generate more false positive calls In general it is recommended that at least one million mammalian cells are required for each IP fraction as defined above For example a 10 cm culture dish of HeLa cells at 70 85 confluence contains approximately 4 to 6 million cells This amount of cells is sufficient for three up to five ChIP fractions after carefully harvesting the cell lysate following cross linking step NOTE 7he actual amounts of cell or tissue samples required to produce high quality quantitative results may need to be determined empirically for each experiment Once determined this amount should be applied uniformly for all samples within an experiment for a consistent performance 2 Cross linking and Chromatin Preparation Follow the recommendation in your ChIP kit or follow your own protocol For Native ChIP without cross linking using antibodies of interest or preparation of enzyme digested chromatin follow the instructions in the related protocols 3 Chromatin DNA Fragmentation by So
16. IP assay and perform the chromatin immuno precipitation using our ChampionChIP One day Kit and Antibody Kits for optimal results Or you can follow another methodology of your choice Subsequently mix your purified ChIP DNA with SABiosciences instrument specific and ready to use RT2 PCR Master Mixes and aliquot the mixture into a ChIP Array plate of your choice Finally perform PCR and determine the level enrichment of your ChIP DNA samples by analyzing Technical Support support SABiosciences com www SABiosciences com 4 Version 1 1 your Real Time PCR raw data with our Excel based ChIP PCR Array Data analysis template The whole procedure can be easily completed in one or two days The streamlined ChampionChIP PCR Array quickly delivers a simple and robust approach for the profiling of a number of ChIP DNA samples with Real Time qPCR precision The reliability and simplicity of the ChIP PCR Array makes this technology feasible for routine use in any research laboratory Benefits of the ChampionChIP PCR Arrays Function or Disease Focused arrays represent a panel of genomic sites relevant to a biological function or disease state Reliable Sensitive amp Specific simple and streamlined procedure for simultaneous analysis of multiple genomic sites with Real Time qPCR precision and sensitivity Feasible to Routine Use brings ChIP profiling on a large set of samples to almost any lab with a Real Time PCR instrument High
17. PCR Array Technical Support 888 503 3187 US 301 682 9200 3 ChampionChIP PCR Arrays I Background and Introduction In eukaryotic cells chromatin is a dynamic complex of DNA RNA and proteins that makes up chromosomes The basic unit of chromatin is the nucleosome which is composed of 147 base pairs of DNA wrapped around an octamer of four core histones H2A H2B H3 and H4 Chromatin in a non dividing cell can be divided into two functional states eurochromatin or heterochromatin Eurochromatin is often but not always enriched with genes in active transcriptional states and plays a central role in the regulation of gene expression Conversely heterochromatin primarily consists of repetitive sequences and repressed genes associated with morphogenesis or differentiation imprinting and X chromosome inactivation Heterochromatin is also a prevalent chromatin state among eukaryotes which has crucial functions in controlling chromosomal stability and the prevention of DNA mutations and translocations Histones are subjected to several types of modifications including acetylation methylation and phosphorylation These modifications change dynamically and co ordinately on multiple genomic sites to regulate gene expression For example in activated gene regions there is an enrichment of modified histones such as H3ac H4ac H3K4me2 or H3K4me3 whereas the enrichment of modified histones H3K9me3 H3K20me3 or H4K27me3 are implicated
18. Real Time PCR Detection Sample 1 Sample 2 112 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 8 19 20 21 22 23 24 7o O zz x n o m 9 o m 7o o z r n o m 9 o m 1 2 3 4 5 6 7 8 9 10 11 12 23 14 15 16 17 18 19 20 21 22 23 24 Sample 4 112 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 O z r p n Pn O m gt Beo r A iz G5 n Pn Oo S m 412 13 415 6 7 8 10 47 2 13 14 15 16 17 18 19 20 21 22 23 24 Figure 6 Loading Cocktail into a 384 well PCR Array To load a 384 well format PCR Array add 10 uL of the Experimental Cocktail from each numbered sample into the staggered wells with the same number as indicated in the figure Technical Support support SABiosciences com www SABiosciences com 20 Step 3 Performing Real Time PCR Detection Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your Real Time instrument Version 1 1 a CAREFULLY but tightly seal the PCR Array with the optical thin wall 8 cap strips Formats A and D or with the optical adhesive film Formats C E F and G b Centrifuge the plate for 1 full minute at room temperature at 1000 g to remove air bubbles Visually inspect the plate from underneath of the plate to ensure no bubbles are
19. Support 888 503 3187 US 301 682 9200 29 ChampionChIP PCR Arrays NOTES Technical Support support SABiosciences com www SABiosciences com 30 Version 1 1 NOTES Technical Support 888 503 3187 US 301 682 9200 31 ChampionChIP PCR Array User Manual Part 1051A Version 1 1 10 20 2009 f SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 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 supporte SABiosciences com
20. e to the inverse proportional relationship between the threshold cycle C and the original amount of template DNA as well as the doubling of the amount of product with every cycle the original amount of template DNA L in each ChIP fraction is expressed as L 2 To control for variation from ChIP fraction to ChIP fraction the amount of each promoter region of interest in the initial chromatin fraction Input provides a convenient normalization factor by establishing the number of genome equivalents present in each initial chromatin fraction Because the Input DNA fraction only represented one percent of the total material the Input C value is adjusted for this dilution factor by subtracting 6 6 cycles The ratio of specific IP over Input provides the fraction of the nuclear factor sites pulled down by the IP target factor Divide the amount of DNA template in the IP fraction by the amount of DNA in the Input fraction to determine the fraction of sites recovered in the IP fraction 9 CP 9 Tett P C Input _ gea ics 2 Ginpur Fraction of Input To determine fold change in fraction enrichment caused by the experimental conditions and nuclear factor activation the normalized amount of the nuclear factor s of interest at the promoter region s of interest in the experimental sample is divided by the corresponding normalized amount in the control sample 9 Atilexpt PI M Where AAC is equal to AC expt AC control Technical
21. erves as reference control for Real Time PCR performance and data normalization IP DNA Genomic DNA purified from IP fraction after immunoprecipitation with an antibody of interest or NIS IgG Control Fraction IgG NIS Mock Fraction The fraction of chromatin mixed with normal IgG or NIS that serves as negative control for ChIP grade antibodies Normal IgG or NIS pulls down non specific DNA providing a measurement of the background or noise for the ChIP system Antibody Fraction The fraction of chromatin mixed with the antibody of interest to pull down its associated genomic DNA sites This provides an estimation of the number of specific genomic sites bound or associated with the nuclear factor of interest in vivo Technical Support 888 503 3187 US 301 682 9200 13 ChampionChIP PCR Arrays ChIP DNA or ChlP DNA Sample Input DNA or IP DNA used for PCR or other downstream analysis 1 Starting Material for Biological Experiment The ChIP PCR Array yields reliable results with as little as one million cells per array plate However the optimal amount of starting material depends on the relative abundance of the protein associated DNA and the enrichment efficiency of the ChIP assay with the antibody of choice Low amounts of starting material will affect the efficiency of the ChIP enrichment The high abundance of the protein associated DNA immunoprecipitated with highly efficient antibodies such as anti H3K4me3 H3K4me2 or
22. es not have the adaptive baseline function you will need to set the baseline manually Use the Linear View of the amplification plots to determine the earliest visible amplification Set the instrument to use the readings from cycle number two 2 through two 2 cycles before the earliest visible amplification but no more than cycle 15 The earliest amplifications usually will be visible between cycles 14 and 18 2 Threshold Fluorescence Value Selection Manually define the Threshold Value by using the Log View of the amplification plots and place it above the background signal but within the lower one third to lower one half of the linear phase of the amplification plot IMPORTANT Ensure that the thresholds are the same across all ChIP PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays If the ChIP DNA sample quality has been adequately controlled the cycling program has been executed properly and the thresholds have been defined correctly then the Ct value of the Positive PCR PPC should be 20 2 across all of your arrays or samples If not see the Troubleshooting and FAQ section 3 Real Time PCR Data Read out Technical Support support SABiosciences com www SABiosciences com 22 Version 1 1 Export the resulting C values for all wells from your instrument to a blank Excel spreadsheet and save for use with the SABiosciences ChampionChIP PCR Array
23. et provided in the Antibody kit If you follow your own protocol and or use another antibody of interest you might find the following considerations useful ChIP Preparation Definitions used in ChampionChIP System See Figure 1 on Page 6 ChIP Sample Starting material for ChIP assay tissue or cells NOTE ChIP can be performed with a single ChIP sample detection of protein DNA association in one condition or multiple samples dependent on experimental design such as knockout vs wild type treated vs untreated or a time course ChIP Ready Chromatin Prepared chromatin after fragmentation step ready for immunoprecipitation process IP Fraction ChIP Ready Chromatin from each ChIP sample must be diluted pre cleared and divided into equal aliquots which are called IP fractions Therefore an IP Fraction represents the fraction of diluted and pre cleared ChIP Ready Chromatin used for the actual immunoprecipitation step with antibody of interest control antibody normal IgG or non immune serum NIS NOTE One ChIP sample is sufficient for two or more IP fractions Input Fraction or Input As an input control 1 of IP Fraction from each ChIP Sample is set aside before the Chromatin Immunoprecipitation step NOTE One ChIP sample needs only one Input control Input DNA Genomic DNA directly purified from Input fraction It provides a measurement of the total number of interested genomic site s included into the IP fraction and s
24. f l SABiosciences 7775 7 WV Biomol www biomol de Phone 49 40 8532600 or 0800 2466651 D U S e r M a n u a Fax 49 40 85326022 or 0800 2466652 D ChampionChIP PCR Array Real Time PCR Based Pathway or Disease Focused Profiling of Chromatin Immunoprecipitation ChIP Samples See Purchaser Notification for limited use license and warranty information page 3 Part 1051A Version 1 1 10 20 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA ChampionChIP PCR Array Real Time PCR Based Pathway or Disease Focused ChIP DNA Profiling User Manual For Catalog Numbers Prefixed by GAH GAM Ordering and Technical Service Contact Information BIOMOL GmbH Waidmannstr 35 C 22769 Hamburg b info biomol de lomo www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information MasterCard Shipping address Billing address For more information visit us at www biomol de SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA Version 1 1 CONTENTS l Background and Introduction 4 I Materials Provided 7 Ill Additional Materials Required 8 IV Protocol 9 A
25. fication highly efficiently Therefore the user will obtain high yields and high quality ChIP DNA free of inhibitors greatly facilitating Real Time PCR profiling ChIP Grade Antibodies are a critical component of the system in order to ensure a reliable ChIP PCR Array profiling The ChampionChIP Antibody Kits provide ChIP Grade Antibodies that have been rigorously validated for specificity and efficiency of genomic DNA enrichment associated with specific transcription factors modified or unmodified histones or other nuclear factors The positive and negative control PCR primer set included in each antibody kit guarantee a successful enrichment of specific genomic loci The positive control PCR primer set detects enrichment of well characterized binding site for the relevant nuclear factor An optimization of the Immunoprecipitation conditions will be required if you follow your own protocol and or use a specific antibody of interest In addition a quantitative analysis of ChIP enriched DNA used for ChIP PCR Array will have to be performed Further details can be found in the ChIP Preparation and Quality Control section page 13 Technical Support support SABiosciences com www SABiosciences com 12 Version 1 1 A ChIP Preparation and Quality Control For ChIP Preparations and Quality control follow instructions specify in the user manual included in the ChampionChIP One Day Kit and ChampionChIP Antibody Kits using the primer s
26. hIP DNA samples which affect the PCR amplification of the positive control will also affect the PCR amplification for the ChIP DNA samples 1 The average Ct value of PPC should be 20 2 on each PCR Array and should not vary by more than two cycles between PCR Arrays 2 The differences in average Ct PPC values between samples indicate the presence of different amounts of PCR amplification inhibitors in each ChIP DNA sample 3 An average PPC C value consistently greater than 22 for all of your samples may indicate a problem with the cycling conditions or may simply be indicative of the relative sensitivity of your instrument See the Troubleshooting and FAQ section Reliability of ChIP PCR Array 1 Genomic DNA Input Control IGX1A Within the right PPC Ct range the IGX1A Ct value of the Input fraction from each experimental sample should be 27 5 1 assuming that the experiment was set up with one million cells a If the IGXA Ct value is 30 it usually indicates that the amount of starting chromatin for the ChIP Assay was too low and or that the recovery efficiency of ChIP DNA from the reverse cross linking and DNA purification is too low This may result in a decreased number of positive calls and increased false negative calls See page 13 ChIP Preparation If the IGXA Ct value is 24 it usually indicates that too much starting material for the ChIP Assay was used which may cause an increased background and false
27. method This is valid because all Ct values for one genomic site are determined from the same PCR primer assay unlike mRNA measurements which are normalized with various PCR primer assays To account for chromatin preparation differences between samples input normalized values for every IP fraction should be used for the calculation of genomic site occupancy When the C values of the Control reactions Control NIS or Mock IP are gt 35 cycles the reproducibility by qPCR is low due to the high level of variation introduced by stochastic effects on the inverse exponential function of PCR in this low template concentration range Thus the Fold Enrichment values from ChIP PCR do not provide a reliable Technical Support support SABiosciences com www SABiosciences com 24 Version 1 1 measure for cross sample comparison Therefore SABiosciences recommends using input normalized values Input Enrichment for all calculations Step by step analysis 1 To account for chromatin sample preparation differences normalize each IP DNA C value to its Input DNA C value for the same PCR Assay in every ChIP sample AC Normalized IP C IP C Input Logs Input dilution factor The default Input dilution Factor is 196 which corresponds with dilution factor of 100 or 6 644 cycles i e log2 of Calculate the 96 Input Before Normalized Enrichment for each IP DNA linear conversion of the Normalized IP AC Input
28. mplies that no inhibitors are present in the ChIP DNA preparations Since the fold enrichment reflected by the C difference is no significant variation among the samples from different volumes the results also demonstrate the reliability of the ChampionChIP System B Performing Real Time PCR Important considerations before starting PCR reaction e For consistent results use the same Real Time PCR instrument to generate all the data within a single experiment e The use of SABiosciences RT Real Time SYBR Green PCR Master Mixes is absolutely critical for obtaining accurate results from the ChIP PCR Array Be sure to use the correct master mix for your instrument before continuing with this protocol See Pages 9 and 10 e The use of the correct ChIP PCR Array plate format is also critical to the success of this experiment Be sure that you have the correct PCR Array format for your instrument before continuing with this protocol See Page 6 e The accuracy and precision of your pipetting will determine the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure not to introduce any bubbles into the wells of the PCR Array Spin plate down before starting PCH e Do not use DEPC treated H2O Use high quality nuclease free H2O If you are not sure whether you re RNase DNase free water has been DEPC treated please check with the supplier Technical Support supp
29. nication For the best balance between resolution sensitivity and IP efficiency it is recommended to fragment the chromatin DNA to an average size between 500 and 1500 base pairs as judged by agarose gel electrophoresis As shown in Figure 3 the reverse cross linking and genomic DNA isolation are required for validation of the ChIP fragments Follow the recommendations specified in your ChIP kit or follow your own protocol The size of chromatin fragments sheared by an enzyme should be controlled as well to ensure good results during the PCR amplification step Technical Support support SABiosciences com www SABiosciences com 14 Version 1 1 Lane 1 000 bp 500 bp Figure 3 Characterization of Chromatin Fragmentation The fast cross linking reverse method provided in the ChampionChIP One Day Kit permits the analysis of sonicated samples by agarose gel electrophoresis in less than 1 hour Lanes 1 1 0 kb ladder 2 Chromatin before sonication 3 Sonicated chromatin after reverse cross linking and DNA extraction following the ChampionChIP One Day Kit User Manual 4 Sonicated chromatin before reverse cross linking and DNA extraction 4 Immunoprecipitation Multiple IP Fractions per Sample The ChIP Ready Chromatin from one ChIP sample will generate the following IP fractions Input Fraction One Input fraction for each ChIP sample experimental sample IP fraction Two or more IP fractions for each ChIP sample
30. nstead 95 C 1 min 65 C 2 min OPTICS OFF 65 C to 95 C at 2 C min OPTICS ON NOTES 1 If you decide not to obtain the dissociation curve immediately save the plates wrapped in aluminum foil at 20 as is in case you need to perform this operation at a later point in time for troubleshooting purposes When ready simply warm the plate to room temperature place it into your Real Time instrument and run the melting program described above 2 Be sure to visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed make a note of which wells so that you may qualify your data analysis appropriately DO NOT open any previously run and stored PCR Array plate Removing the thin wall 8 cap strips or the adhesive film from PCR Arrays releases PCHR product DNA into the air where it will contaminate and confound the results of future Real Time PCR experiments See also the Note on Preparing a Workspace Free of DNA Contamination Technical Support 888 503 3187 US 301 682 9200 23 ChampionChIP PCR Arrays D ChIP PCR Array Data Analysis ChampionChIP PCR Array Data Analysis The Excel based ChIP PCR Array Data Analysis template provides a guideline and template for automatically performing the calculations and interpretation of the control wells upon including threshold cycle data from a Real Time PCR instrument The ChIP PCR Array Data Analysis tool present
31. ody Kit Therefore the use of the complete ChampionChIP system is absolutely essential for obtaining optimal Real Time PCR profiling results PCR Reagents The chemically modified and tightly controlled HotStart enzyme and other proprietary chemical components in our RT qPCR Master Mixes ensure accurate SYBR Green based PCR results by preventing the formation of primer dimers and other nonspecific PCR products They are also helpful in ensuring the high amplification efficiencies even for Technical Support 888 503 3187 US 301 682 9200 11 ChampionChIP PCR Arrays genomic regions that are difficult to amplify When we test other sources of enzymes with our PCR Arrays we frequently saw primer dimers and other non specific products that confound SYBR Green based Real Time PCR detection Because each instrument uses a different reference dye to normalize their optics make sure that you select the correct Master Mix for the Real Time PCR instrumentation at your laboratory Chromatin Immunoprecipitation ChIP The results from the ChIP PCR Array system are based on the efficient enrichment of specific genomic loci using ChIP Assay The ChampionChIP One Day Kit is a simple and robust ChIP preparation kit It works with an optimized Immunoprecipitation buffer system to improve efficiency and specificity of antibody binding to chromatin More importantly the kit is specially developed to perform the reverse cross linking and DNA puri
32. ort SABiosciences com www SABiosciences com 18 Version 1 1 Step 1 Sample Preparation Mix the following components in a 5 ml tube or a multi channel reservoir Plate Format 96 well 384 well ACD amp F E amp G 2X SABiosciences RT qPCR Master Mix 1275 yu 550 ul ChIP DNA Sample 180 ul 100 ul DNase free ddH O 1095 ul 450 ul Total Volume 2550 ul 1100 ul NOTES 1 2 This recipe provides an excess volume of ONLY 150 uL 96 well format or 140 UL 384 well format per ChIP DNA sample Very carefully add the cocktail to the PCR Array precisely as described below to insure that each well receives the required volume Save the remainder of the ChIP DNA at 20 C for troubleshooting purposes Step 2 Loading for the 96 Well PCR Arrays Formats A B C and D Please select your PCR Array Format for loading instructions Loading the 96 Well PCR Array Formats A C D or F a b C CAREFULLY remove the PCR Array from its sealed bag Add 25 uL of the Experimental Cocktail to each well of the PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips Change pipet tips following each addition to avoid any cross contamination between the wells or reactions Proceed to the next section STEP 3 on Performing Real Time PCR Detection Loading for the 384 Well PCR Array Format E Each 384 well plate characterizes four samples in separa
33. present in each well Bubbles remaining in the bottom of the wells of a PCR Array will interfere with results c Place the plate on ice while setting up the PCR cycling program below d Place one plate in your Real Time thermal cycler If recommended by your instrument s user manual use a compression pad with the optical film sealed plate formats NOTE ChIP PCR Arrays containing experimental cocktail may be stored at 20 C wrapped in aluminum foil for up to one week until ready to run e Enter and run the appropriate program for your Real Time instrument below If prompted by your instrument software select Absolute Quantitation to begin NOTE For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarrayprotocolfiles php Use a two step cycling program for the following instrumentation ABI 5700 7000 7300 7500 7700 7900HT BioRad iCycler iQ5 MyiQ Stratagene Mx3000p Mx3005p Mx4000p Eppendorf Mastercycler ep realplex Cycles Duration Temperature 10 minutes 95 C 15 seconds 95 C di 1 minute 60 C Use an extended two step cycling program for the following instrumentation Roche LightCycler 480 Cycles Duration Temperature 1 10 minutes 95 C 15 seconds 95 C 1 minute 60 C Attention Roche LightCycler 480 Users Adjust the ramp ra
34. red properly at 20 C The ChIP PCR Arrays are available in six different plate formats each tailored to a specific subset of Real Time PCR instruments and block format Formats A C D and F are for 96 well plates while Formats E and G are for 384 well plates Format For Real Time Instruments Plate ABI standard blocks 5700 7000 7300 7500 7700 7900HT 96 block Bio Rad iCycler iQ5 MyiQ Chromo4 MJ Research A Eppendorf MasterCycler ep RealPlex Sese Stratagene Mx3005p Mx3000p Takara TP 800 C ABI 7500 FAST block 7900HT FAST block StepOnePlus 96 well Bio Rad CFX96 Opticon and Opticon 2 MJ Research D Stratagene Mx4000 96 well ABI 7900HT 384 well block i E Bio Rad CFX384 Been F Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 well block 384 well NOTE 1 The format of the ChIP PCR Array is indicated by the last digit of the catalog number Confirm that you have the correct ChIP PCR Array format for your instrument before starting the experiment 2 The 96 well ChIP PCR Arrays Formats A C D and F are shipped in sets of four 4 twelve 12 or twenty four 24 while the 384 well ChIP PCR Arrays Formats E and G are shipped in sets of Two 2 Four 4 or twelve 12 3 Each ChIP PCR Arrays shipment includes the arrays and either twelve 12 optical thin wall 8 cap strips Formats A and D or one 1 optical adhesive film Formats
35. s the results in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included Download our free Excel based ChlP PCR Array Data Analysis template available for 96 well and 384 well format at http sabiosciences com chipqpcrdataanalysis php Excel Based ChIP PCR Array Data Analysis 1 Download the Excel based ChIP PCR Array Data Analysis Templates by Clicking on the above URL 2 Save and rename the Excel file to your local computer Paste in your Ct values and the results will be generated automatically 3 View and analyze the results by simply following the guidelines specified in the Instruction worksheet of the Excel template NOTE The ChIP PCR Array Data Analysis Template automatically changes all Ct values reported as greater than 35 or as N A not detected to 35 At this point any Ct value equal to 35 is considered a negative call How to Calculate the Percentage Enrichment ChIP results are reported for three levels of genomic site occupancy depending on the experimental question s asked 9e Input Enrichment Fold Enrichment and Fold Difference Differential Occupancy The first two are specific characteristics of a single sample while the Fold Difference Differential Occupancy is a comparison across multiple samples from different experimental conditions When using Real Time PCR for the analysis of ChIP DNA samples all results can be calculated using the AAC
36. t of the genomic DNA by ChIP a The closer the C Ab values equal C Con the less enrichment is achieved b The C Ab average may be used to evaluate the enrichment over the panel of genomic sites on the Array c Most importantly if Input Ab gt 0 1 and or Input Ab Input Con gt 0 01 it indicates possible enrichment If Input Ab lt 0 1 and or Input Ab Input Con 0 01 it indicates no enrichment with that specific antibody NOTE The ChIP PCR Array Data Analysis Template automatically replaces all subtraction values Input Ab Input Con reported as less than 0 01 or as N A not detected to 0 01 At this point the value of any Input Ab that equals to 0 01 is considered as a negative call in other words no enrichment Technical Support 888 503 3187 US 301 682 9200 21 ChampionChIP PCR Arrays VI Troubleshooting and FAQs Please check the QC Report worksheet of ChampionChIP PCR Data Analysis Template for the details If you have additional questions please check our website www sabiosciences com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 1 888 503 3187 or 301 682 9200 Technical Support support SABiosciences com www SABiosciences com 28 Version 1 1 Appendix AACt Data Analysis Method amp Bibliography Detailed Mathematical Explanation of AACt Data Analysis Method Du
37. te sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multi channel pipettors will allow you to properly skip rows or columns when adding each sample Be sure to load each sample into the correct set of wells Use Figure 6 as a guide a b CAREFULLY remove the PCR Array from its sealed bag Load sample cocktails to appropriate wells of the PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips using the provided 384EZLoad Covers Catalog PA 384 and the figure below as a guide Technical Support 888 503 3187 US 301 682 9200 19 ChampionChIP PCR Arrays e Place Cover 1 white on the plate Add 10 uL of Sample 1 cocktail to the open wells Odd number wells of rows A C E G I K M amp O Remove amp discard the cover e Place Cover 2 yellow on the plate Add 10 uL of Sample 2 cocktail to the open wells Even number wells of rows A C E G I K M amp O Remove amp discard the cover e Place Cover 3 black on the plate Add 10 uL of Sample 3 cocktail to the open wells Odd number wells of rows B D F H J L N amp P Remove amp discard the cover Place Cover 4 red on the plate Add 10 uL of Sample 4 cocktail to the open wells Even number wells of rows B D F H J L N amp P Remove amp discard the cover C Proceed to the next section STEP 3 on Performing
38. te to 1 C sec Please refer to the Instrument Setup Guide at http sabiosciences com pcrarrayprotocolfiles php for more information on other REQUIRED changes to settings for Melt Curve Acquisition Technical Support 888 503 3187 US 21 301 682 9200 ChampionChIP PCR Arrays Use a three step cycling program for the following instrumentation ABI StepOnePlus Cycles Duration Temperature BioRad CFX96 CFX384 Opticon 1 10 minutes 95 C Opticon 2 Chromo 4 MJ Research 15 seconds 95 C Takara TP 800 30 to 40 All other instruments 40 seconds 9 9 30 seconds 72 C 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 Different instruments need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 55 C for your instrument C PCR Data Acquisition amp Quality Assessment Calculate the threshold cycle Ct for each well using the instrument s software Please consult the instrument user manual or contact the manufacturers technical support department for further directions 1 Baseline Cycle Range Selection To define the Baseline choose the Automated Baseline option if your instrument has the Adaptive Baseline Function check with instrument manual or manufacturer if unsure If it do
39. terns associated with either transcriptionally active euchromatin or transcriptionally inactive euchromatin or heterochromatin SAbiosciences provides a panel of Real Time PCR primers for a complete and reliable analysis of the histone modifications that regulate chromatin structure and influence gene activity Figure 4 The results of the ChIP Array are dependent on the quantity of DNA enriched by the ChIP Assay In particular the amount of enriched DNA at specific genomic loci is directly related to the specificity and efficiency of the antibody used It is well known that many modified histone markers have distinct distribution patterns associated with transcriptional active euchromatin transcriptional inactive euchromatin or heterochromatin For a reliable analysis of the histone modifications distribution a control panel of Real Time PCR primers was developed targeting different genomic loci associated with transcriptional active inactive and silenced genomic regions With these controls the specificity and efficiency of ChIP DNA enrichment can be reliably analyzed Figure 4 For details please check our website http www sabiosciences com chipgradeantibody php Technical Support support SABiosciences com www SABiosciences com 16 Version 1 1 25 Active Gene Inactive Gene Heterochromatic Region k e 20 GAPDH SAT2 E RP30 3 MYOD1 Sata c ALDOA E SERPINA m IGX1A Percent Input H3K4me3 H3K27me3 H3K9me3 Control IgG
40. vious experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment free of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and lab ware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipette barrels tube racks etc before each new experiment with UV light or 1096 bleach to render any contaminating DNA ineffective in PCR Ultraviolet light will induce the formation of thymidine dimers and bleach will chemically inactivate and degrade any DNA 3 Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any lab ware tips or tubes containing PCR products or other DNA treat these items with 1096 bleach 4 Do not leave lab ware tubes and tip boxes exposed to the air for long periods of time 5 Do not open any post PCR tubes plates in the PCR setup working area Opening tubes plates after the run can release PCR products into the air which will contaminate and confound the results of future Real Time PCR experiments Master Mix and Other ChIP related Reagents Considerations The performance of our ChIP PCR Arrays is only guaranteed with SABiosciences RT qPCR Master Mixes ChampionChIP One Day Kit and the ChampionChIP Antib
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