MethylFlash ™ Methylated DNA Quantification Kit
Contents
1. MethylFlash Methylated DNA Quantification Kit Fluorometric Base Catalog P 1035 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The MethylFlash Methylated DNA Quantification Kit Fluorometric is suitable for detecting global DNA methylation status using DNA isolated from any species such as mammals plants fungi bacteria and viruses in a variety of forms including but not limited to cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples and body fluid samples This kit is particularly suitable for samples only available in small amounts such as laser capture microdissection samples and embryos Input DNA The amount of DNA for each assay can be 20 200 ng For optimal quantification the input DNA amount should be 100 ng as methylated DNA varies from tissue to tissue and can be less than 1 of total DNA in some species Starting Materials Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples body fluid samples etc Internal Control Both negative and positive DNA controls are provided in this kit A standard curve can be performed range 0 2 to 10 ng or a single quantity of methylated DNA can be used as a positive control Because global methylation can vary from tissue to tissue and from normal and diseased states it is advised to run replicate sample2. 09 22 P 1035 Incubator for 37 C incubation Plate seal or Parafilm M Distilled water 1X TE buffer pH 7 5 to 8 0 1X PBS pH 7 2 to 7 5 Isolated DNA of interest Oo Oo Oo o O o GENERAL PRODUCT INFORMATION Quality Control Each lot of the MethylFlash Methylated DNA Quantification Kit Fluorometric is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The MethylFlash Methylated DNA Quantification Kit Fluorometric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The MethylFlash Methylated DNA Quantification Kit Fluorometric and methods of use contain
3. Frame 6 12 4 C User Guide 1 1 RT Spin the solution down to the bottom prior to use Note The MF3 Negative Control is an unmethylated polynucleotide containing 50 of cytosine The MF4 Positive Control is a methylated polynucleotide containing 50 of 5 methylcytosine SHIPPING amp STORAGE The kit is shipped in three parts the first part at ambient room temperature and the second and third parts on frozen ice packs at 4 C Upon receipt 1 Store MF3 MF4 MF6 MF7 and MF8 at 20 C away from light 2 Store MF1 MF5 MF9 and 8 Well Assay Strips at 4 C away from light 3 Store MF2 and MF10 at room temperature away from light All components of the kit are stable for 6 months from date of shipment when stored properly Note Check MF1 10X Wash Buffer contains salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED oO oO o m Adjustable pipette multi channel recommended Aerosol resistant pipette tips Fluorescence microplate reader capable of reading fluorescence at excitation 530 and emission 590 1 5 ml microcentrifuge tubes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2014
4. Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 c 5 hme Unmethylated DNA Methylated DNA Hydroxymethylated DNA T C G T C G A C G T C G T C G A C G T C G T C G A C G The broader functions of 5 hmC in epigenetics are still a mystery today However a line of evidence does show that 5 hmC plays a role in DNA methylation structures and patterns Because of the presence of both 5 mC and 5 hmC in DNA with possibly different functions it is important to determine the contents of these two modified nucleotides and their ratios in different cell types and in different compartments of the genome of mammaiians It is particularly important to identify that in healthy and diseased human cell tissues the epigenetic change at the DNA level is due to methylation or hydroxymethylation Several chromatography based techniques such as HPLC TLC mass spectrometry are used for detecting 5 mC and 5 hmC However these methods are time consuming and have low throughput with high costs To address this problem Epigentek offers the Methy Flash Methylated DNA Quantification Kit Fluorometric to quantify 5 mC or methylated DNA This kit is optimized for paired use with our Methy Flash Hydroxymethylated DNA Quantification Kit for simultaneously quantifying
5. same day 2 Preparation of Diluted Positive Control MF4 Single Point Control Preparation Dilute MF4 Positive Control with 1X TE to 5 ng ul ex 1 ul of MF4 3 ul of TE Representative Standard Curve Preparation First dilute MF4 to 10 ng ul ex 5 ul of MF4 5 ul of 1X TE Then further prepare five different concentrations with the 10 ng ul diluted MF4 and 1X TE into 0 5 1 0 2 0 5 0 and 10 0 ng ul according to the following dilution chart Resulting MF4 Tube MF4 10 ng pl 1XTE Concentration 1 1 0 ul 19 0 ul 0 5 ng ul 2 1 0 ul 9 0 ul 1 0 ng ul 3 1 0 ul 4 0 ul 2 0 ng ul 4 2 0ul 2 0ul 5 0 ng l 5 4 0 ul 0 0 ul 10 0 ng ul 3 DNA Binding a Predetermine the number of strip wells required for your experiment Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Add 80 ul of MF2 Binding Solution to each well c Add 1 ul of MF3 1 ul of Diluted MF4 see note below and 100 ng of your Sample DNA 1 8 ul into the corresponding wells as shown in Table 1 or Table 2 Mix solution by gently tilting from side to side or shaking the plate several times Ensure the solution coats the bottom of the well evenly Note 1 For a single point control add 1 ul of MF4 at a concentration of 5 ng ul as prepared in Step 2 For the standard curve add 1 ul of Diluted MF4 at concentrations of 0 5 to 10 ng ul see the c
6. 0 as the positive control contains only 50 of 5 mC Example calculation Average RFU of MF3 is 1000 Average RFU of MF4 is 31000 Average RFU of Sample is 21000 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 S is 100 ng P is 5 ng 21000 1000 100 5 mC x 100 1 673 31000 1000 XZ 5 Absolute Quantification To quantify the absolute amount of methylated DNA using an accurate calculation first generate a standard curve and plot the RFU values versus the amount of MF4 at each concentration point Next determine the slope RFU ng of the standard curve using linear regression Microsoft Excel s linear regression functions are suitable for such calculation and the most linear part at least 4 concentration points including 0 point of the standard curve for optimal slope calculation Now calculate the amount and percentage of methylated DNA 5 mC in total DNA using the following formulas Sample RFU MF3 RFU nie N ANT Slope x 2 5 mC Amount ng 5 mC X 100 S S is the amount of input sample DNA in ng 2 is a factor to normalize 5 mC in the positive control to 100 as the positive control contains only 20 of 5 mC Example calculation Average RFU of MF3 is 1
7. 000 Average RFU of sample is 21000 Slope is 6000 RFU ng S is 100 ng 21000 1000 5 mC ng 1 67 ng 6000 x 2 1 67 x 100 1 67 100 5 mC o I SUGGESTED STRIP WELL SETUP Table 1 The suggested strip well plate setup using a single point positive control in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A MF3 MF3 Sample Sample Sample Sample B MF4 MF4 Sample Sample Sample Sample c Sample Sample Sample Sample Sample Sample D Sample Sample Sample Sample Sample Sample E Sample Sample Sample Sample Sample Sample 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 F Sample Sample Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample Table 2 The suggested strip well plate setup for standard curve preparation in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well St
8. NA contains 50 of cytosine and methylated DNA contains 50 of 5 methylcytosine were added into the assay wells at different concentrations and then measured with the MethylFlash Methylated DNA Quantification Kit Fluorometric For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input DNA Amount DNA amount can range from 20 ng to 200 ng per reaction An optimal amount is 100 ng per reaction Starting DNA may be in water or in a buffer such as TE DNA Isolation You can use your method of choice for DNA isolation Epigentek offers a series of genomic DNA isolation kits for your convenience DNA Storage Isolated genomic DNA can be stored at 4 C short term or 20 C long term until use 1 Preparation of 1X Wash Buffer MF1 48 Assay Kit Add 13 ml of MF1 10X Wash Buffer to 117 ml of distilled water pH 7 2 7 5 96 Assay Kit Add 26 ml of MF1 10X Wash Buffer to 234 ml of distilled water pH 7 2 7 5 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 Note This Diluted MF1 1X Wash Buffer can now be stored at 4 for up to six months All other diluted solutions should be kept on ice at all times and should be discarded if not used within the
9. both methylated and hydroxymethylated DNA or for quantifying methylated DNA by itself The kit has the following advantages and features e Fluorometric assay with easy to follow steps for convenience and speed The whole procedure can be finished within 4 hours e Innovative kit composition enables background signals to be extremely low which eliminates the need for plate blocking and allows the assay to be simple accurate reliable and consistent e High sensitivity of which detection limit can be as low as 50 pg of methylated DNA e Optimized antibody and enhancer solutions allow high specificity to 5 mC with no cross reactivity to unmethylated cytosine and no or negligible cross reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA e Universal positive and negative controls are included which are suitable for quantifying methylated DNA from any species e Strip microplate format makes the assay flexible manual or high throughput analysis References Robertson KD Nat Rev Genet 6 597 610 2005 Kriaucionis S et al Science 324 929 930 2009 WYATT GR et al Biochem J 55 774 8 1953 Tahiliani M et al Science 324 930 935 2009 Valinluck V et al Nucleic Acids Res 32 4100 4108 2004 Valinluck V et al Cancer Res 67 946 50 2007 Jin SG et al Nucleic Acids Res 38 e125 2010 Quon RNs PRINCIPLE amp PROCEDURE The MethylFlash Methylated DNA Quantification Kit Fluorome
10. erly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly capped after each opening or use No signal or weak signal in only the positive control wells The positive control DNA is insufficiently added to the well in Step 3c Ensure a sufficient amount of positive control DNA is added The MF4 Positive Control is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of MF4 Positive Control 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 9 Printed 2014 09 22 P 1035 High background present in the negative control wells Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by sample or positive control DNA Ensure the well is not contaminated from adding sample or positive control DNA accidentally or from using contaminated tips Incubation time is too long The incubation time at Step 3d should not exceed 2 h Over development of fluorescence Decrease the development time in Step 5b Large variation between replicate wells Fluorescent reaction is not evenly occurring due to an incon
11. hart in Step 2 The final amounts should be 0 5 1 2 5 and 10 ng per well 2 For optimal binding sample DNA volume added should not exceed 8 ul 3 To ensure that MF3 Diluted MF4 and sample DNA are completely added into the wells the pipette tip should be placed into the MF2 solution in the well and aspirated in out 1 2 times d Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min e Remove the MF2 Binding Solution from each well Wash each well with 150 ul of the Diluted MF1 1X Wash Buffer each time for three times This can be done by simply pipetting Diluted MF1 in and out of the wells 4 Methylated DNA Capture a Dilute MF5 at 1 1000 ratio with the Diluted MF1 b Add 50 ul of the Diluted MF5 to each well then cover and incubate at room temperature for 60 min c Remove the Diluted MF5 solution from each well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 m Wash each well with 150 ul of the Diluted MF1 each time for three times Dilute MF6 at 1 2000 ratio with the Diluted MF1 Add 50 ul of the Diluted MF6 to each well then cover and incubate at room temperature for 30 min Remove the Diluted MF6 solution from each well Wash each well with 150 ul of the Diluted MF1 each t
12. ime for four times Dilute MF7 at 1 5000 ratio with the Diluted MF1 Add 50 ul of the Diluted MF7 to each well then cover and incubate at room temperature for 30 min Remove the Diluted MF7 solution from each well Wash each well with 150 ul of the Diluted MF1 each time for five times Wash each well with 150 ul of the 1X PBS one time 5 Signal Detection a Prepare Fluoro Development Solution by adding 1 ul of MF8 and 1 ul of MF9 into each 500 ul of MF10 Add 50 ul of Fluoro Development Solution into the wells and incubate at room temperature for 1 to 4 minutes away from light The color in the standard wells containing the higher concentrations may turn pink during this period Measure and read RFU relative fluorescence units on a fluorescence microplate reader at 530 y 590ey nm If the strip well frame does not fit the microplate reader transfer the solution to a standard 96 well microplate and read the RFU on a fluorescence microplate reader at 530 590 y nm 6 5 mC Calculation Relative Quantification To determine the relative methylation status of two different DNA samples simple calculation for percentage of 5 mC in total DNA can be carried out using the following formula Sample RFU MF3 RFU S 5 mC O _ K 100 MF4 RFU MF3 RFU x 2 P S is the amount of input sample DNA in ng P is the amount of input positive control MF4 in ng 2 is a factor to normalize 5 mC in the positive control to 10
13. o be empty or insufficient in volume Buffer evaporated due to the very small volumes resulting in a higher concentrated antibody Add 1X PBS buffer into the Capture Antibody vial until you restore the correct intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2014 09 22 P 1035 RELATED PRODUCTS DNA Sample Preparation P 1003 FitAmp General Tissue Section DNA Isolation Kit P 1004 FitAmp Plasma Serum DNA Isolation Kit P 1006 DNA Concentrator Kit P 1007 FitAmp Gel DNA Isolation Kit P 1009 FitAmp Paraffin Tissue Section DNA Isolation Kit P 1017 FitAmp Urine DNA Isolation Kit P 1018 FitAmp Blood and Cultured Cell DNA Extraction Kit Global DNA Methylation Quantification P 1034 MethylFlash Methylated DNA Quantification Kit Colorimetric P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric P 1037 MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All
14. proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methyl group at the 5 carbon of the cytosine ring by DNA methyltransferases resulting in 5 methylcytosine 5 mC In somatic cells 5 mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG whereas in embryonic stem ES cells a substantial amount of 5 mC is also observed in non CpG contexts The biological importance of 5 mC as amajor epigenetic modification in phenotype and gene expression has been recognized widely For example global decrease in 5 mC content DNA hypomethylation is likely caused by methyl deficiency due to a variety of environmental influences and has been proposed as a molecular marker in multiple biological processes such as cancer It has been well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer Thus the quantification of 5 mC content or global methylation in cancer cells could provide very useful information for detection and analysis of this disease Quite recently a novel modified nucleotide 5 hydroxymethylcytosine 5 hmC has been detected to be abundant in mouse brain and embryonic stem cells In mammals it can be generated by oxidation of 5 methylcytosine a reaction mediated by the Tet family of enzymes and Dnmt proteins It is a hydroxylated and methylated form of cytosine 110 Bi County Blvd
15. rights reserved Products are for research use only P 1035
16. rip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A MF3 MF3 Sample Sample Sample Sample B MF4 0 5 ng MF4 0 5 ng Sample Sample Sample Sample c MF4 1 ng MF4 1 ng Sample Sample Sample Sample D MF4 2 ng MF4 2 ng Sample Sample Sample Sample E MF4 5 ng MF4 5 ng Sample Sample Sample Sample F MF4 10 ng MF4 10 ng Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Causes Suggestions No signals for both the positive control and samples Reagents are added incorrectly Check if reagents are added in the proper order and if any steps in the protocol may have been omitted by mistake The well is incorrectly washed before DNA binding Ensure the well is not washed before adding positve control and samples The bottom of the wells are not compeleted covered by the MF2 Binding Solution Ensure the solution coats the bottom of the well by gently tilting from side to side or shaking the plate several times Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol is followed correctly Insufficient input materials Ensure that a sufficient amount of positive control and samples are added into the wells Incorrect fluorescence reading Check if appropriate fluorescence wavelength 530 x 590em nm is used Kit was not stored or handled prop
17. s at different DNA concentrations to ensure that the signal generated falls within range of the microplate reader used This kit will allow the user to quantify an absolute amount of methylated DNA and determine the relative methylation states of two different DNA samples Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 1035 48 Cat P 1035 96 Upon Receipt MF1 10X Wash Buffer 14 ml 28 ml 4 C MF2 Binding Solution 5 ml 10 ml RT MF3 Negative Control 20 ug ml 10 ul 20 ul 20 C MF4 Positive Control 20 g ml 10 ul 20 ul 20 C MF5 Capture Antibody 1000 ug ml 4ul 8 ul 4 MF6 Detection Antibody 400 ug ml 8 ul 16 pl 20 C MF7 Enhancer Solution 8 ul 16 ul 20 C MF8 Fluoro Developer 8 ul 16 ul 20 C MF9 Fluoro Enhancer 8 ul 16 ul 4 C MF10 Fluoro Dilutor 4ml 8 ml RT 8 Well Assay Strips With
18. sistency in pipetting time Ensure MF8 Fluoro Developer is added at the same time between replicates or otherwise maintain a consistent timing in between each addition of solutions Fluorescent reaction is not occurring evenly due to an inconsistent order of adding solutions Ensure all solutions particularly MF8 Fluoro Developer are added in the same order each time as all other solutions The solutions are not evenly added due to an inconsistency in pipetting volume Ensure the solution in each pipette tip is equal in the multi channel pipette Equilibrate the pipette tip in any solutions before adding them Ensure the solutions especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not actually added into the wells Do not allow the pipette tip to touch the outer edges or inner sides of the wells in order to prevent solutions from sticking to the surface Did not sufficiently shake the solutions in the wells after adding sample or positive control at step 3c Gently and evenly shake the plate frame across a flat surface so that the solutions in the wells are better distributed Do not stir Did not use the same pipette device throughout the experiment Use the same multi channel pipette device throughout the entire experiment as different pipette devices may have slight variations in performance Capture Antibody vial appears t
19. tric contains all reagents necessary for the quantification of global DNA methylation In this assay DNA is bound to strip wells that are specifically treated to have a high DNA affinity The methylated fraction of DNA is detected using capture and detection antibody and then quantified fluorometrically by reading the RFU relative 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1035 fluorescence units with a fluorescence spectrophotometer The amount of methylated DNA is proportional to the fluorescence intensity measured Prepare genomic DNA We recommend using Epigentek s series of DNA isolation kits Bind DNA to assay well Wash wells then add capture antibody Wash wells then add detection antibody and enhancer solution Add fluoro developing then measure RFU Schematic procedure of the MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric PROTOCOL solution for fluorescence 30000 Methylated DNA 25000 Unmethylated DNA 20000 15000 10000 5000 0 0205 1 2 5 10 100 Input DNA ng Demonstration of high sensitivity and specificity of methylated DNA detection achieved by the MethylFlash kit Synthetic unmethylated D
Download Pdf Manuals
Related Search