EpiQuik ™ 8-OHdG DNA Damage Quantification Direct Kit
Contents
1. EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric Base Catalog P 6004 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric is suitable for detecting oxidative DNA damage 8 OHdG 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 and body fluid samples Input DNA The amount of DNA for each assay can be 100 ng to 300 ng For optimal quantification the input DNA amount should be 300 ng as basal 8 OHdG is generally less than 0 01 of total DNA Starting Material 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 blood body fluid samples etc Internal Control Both negative and positive DNA controls are provided in this kit A standard curve can be performed range 5 to 200 pg of 8 OHdG or a single quantity of 8 OHdG can be used as a positive control Because 8 OHdG content can vary from tissue to tissue and from normal and diseased states or vary under treated and untreated conditions it is advised to run replicate samples to ensure that the signal generated is validated This kit will allow the user to quantify an absolute amount of 8 OHdG and determine the relat2. 100 pg ul 1 pl PC 9 ul TE Suggested Standard Curve Prep First dilute PC to 200 pg ul 2 ul of PC 8 ul of 1X TE Then further prepare 6 different concentrations with the 1 ng ul diluted PC and 1X TE into 5 10 20 50 100 and 200 pg ul according to the following dilution chart Resulting PC Concentration 1 0 ul 39 0 ul 5 pg ul 1 0 ul 19 0 ul 10 pg ul 1 0 ul 9 0 ul 20 pg ul 1 0ul 3 0 ul 50 pg ul 2 0 ul 2 0 ul 100 pg ul 3 0 ul 0 0 ul 200 pg ul Tube PC 200 oe 1 01 an DG ojaa N Note Keep each of the diluted solutions except Diluted WB 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day 3 DNA Binding 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 23 Epigentek Group Inc All rights reserved Products are for research use only P 6004 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 Add 80 ul of BS Binding Solution to each well Add 1 ul of NC 1 ul of Diluted PC see note below and 300 ng of your Sample DNA 1 8 ul into the designated wells depicted in Table 1 or Table 2 Mix solution by gently tilt
3. Page 3 Printed 2014 09 23 P 6004 useful information for the better understanding of oxidative damage disease relationships which benefits diagnostics and therapeutics of the disease can be obtained o N poe sae dey N as eo HN HN N HOHE oO HOH C o on 2 dG OH 4 OH dG Forme f de ne 8 OHdG t en radica Several chromatography based techniques such as HPLC ECD and LC MS are used for detecting 8 OHdG in tissues and cells However these methods are time consuming and have low throughput with high costs The currently used competitive ELISA methods are also not conveniently applicable for cell tissue 8 OHdG detection because they are less accurate and have an inability to use intact DNA isolated from cells or tissues directly To address these problems Epigentek offers the EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric which uses a unique procedure to directly quantify 8 OHdG in cells tissues The kit has the following advantages and features e Fluorormetric assay with easy to follow steps for convenience and speed The entire procedure can be completed within 3 hours and 45 minutes e High sensitivity of which the detection limit can be as low as 1 pg of 8 OHdG e High specificity by detecting only 8 OHdG without cross reactivity to 8 OHdG analogues such as dG guanine 8 OHGua and 8 OHG within the indicated concentration range of the sample DNA e Direct detection of 8 OHdG using intact DNA which elimina
4. 5500 1500 0 1 Absolute Quantification To quantify the absolute amount of 8 OHdG using an accurate calculation first generate a standard curve and plot the OD values versus the amount of PC at each concentration point Next determine the slope OD ng of the standard curve using linear regression Microsoft Excel s linear regression functions are suitable for such calculation and also determine the most linear part include at least 4 concentration points of the standard curve for optimal slope calculation Now calculate the amount and percentage of 8 OHdG in your total DNA using the following formulas Sample RFU NC RFU 8 OHdG ng Slope 8 OHdG Amount 8 OHdG X100 S S is the amount of input sample DNA in ng Example calculation Average RFU of NC is 1500 Average RFU of sample is 2100 Slope is 40000 RFU ng S is 300 ng 2100 1500 8 OHdG ng _ 0 015 ng 40000 0 015 8 OHdG _ x 100 0 005 300 Page 8 Pri 2014 09 2 Epigentek Group Inc All rights reserved Products are for research use only eee P 6004 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 Str
5. 9 23 Epigentek Group Inc All rights reserved Products are for research use only P 6004 2 Buffer and Solution Preparation a Preparation of 1X Wash Buffer 48 Assay Kit Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water pH 7 2 7 5 96 Assay Kit Add 26 ml of WB10X Wash Buffer to 234 ml of distilled water pH 7 2 7 5 This Diluted WB 1X Wash Buffer can now be stored at 4 C for up to six months Prepare Diluted CA Capture Antibody Solution Dilute CA Capture Antibody with Diluted WB at a ratio of 1 100 i e add 1 ul of CA to 100 ul of Diluted WB About 50 ul of this Diluted CA will be required for each assay well Prepare Diluted DA Detection Antibody Solution Dilute DA Detection Antibody with Diluted WB1 at a ratio of 1 1000 i e add 1 ul of DA to 1000 ul of Diluted WB About 50 ul of this Diluted DA will be required for each assay well Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of Diluted WB About 50 ul of this Diluted ES will be required for each assay well Prepare Fluorescence Development Solution Add 1 ul of FD Fluoro Developer and 1 ul of FE Fluoro Enhancer to every 500 ul of DB Dilution Buffer About 50 ul of this Fluorescence Development Solution will be required for each assay well Preparation of Diluted Positive Control Single Point Control Prep Dilute PC Positive Control with 1X TE to
6. cubate at room temperature for 2 to 4 min away from direct light The Fluorescence Development Solution will turn pink in the presence of sufficient 8 OHdG products Read the fluorescence on a fluorescence microplate reader within 2 to 10 min at 530 590 nm Note If the strip well microplate frame does not fit in the fluorescence microplate reader transfer the solution to a standard 96 well microplate 6 8 OHdG Calculation 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 23 P 6004 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 Relative Quantification To determine the relative 8 OhdG status of two different DNA samples a simple calculation for the percentage of 8 OHdG in your total DNA can be carried out using the following formula Sample RFU NC RFU S 8 OHdG X 100 PC RFU NC RFU P S is the amount of input sample DNA in ng P is the amount of input positive control PC in ng Example calculation Average RFU of NC is 1500 Average RFU of PC is 5500 Average RFU of Sample is 2100 S is 300 ng P is 0 1 ng 100 pg 2100 1500 300 8 OHdG ____________ _ x 1003 0 005
7. e 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 EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW 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 8 hydroxy 2 deoxyguanosineis 8 OHdG or 8 oxo dG is an oxidized derivativ
8. e of deoxyguanosine and is generated by hydroxyl radicals singlet oxygen and one electron oxidants in cellular DNA As a modified nucleoside base 8 OHdG is considered important not only because of its abundance but also because of its mutagenic potential through G to T transversion mutations upon replication of DNA 8 OHdG also participates in epigenetic regulation of gene activation repression by inhibiting the binding affinity of MBD proteins to the CpG sites of DNA It is generally accepted that oxidatively damaged DNA can be repaired by base excision repair BER enzymes Currently 8 OHdG is widely accepted as a sensitive marker of oxidative DNA damage and oxidative stress Evidence shows that increased levels of 8 OHdG are closely correlated with exposure to harmful environmental factors such as ionizing radiation industrial chemicals air pollution cigarette smoking and cancer chemotherapy It has also been demonstrated that increased concentrations of 8 OHdG are pathogenically linked to a variety of age associated diseases including cancer coronary heart disease diabetes and neurodegenerative diseases such as Alzheimer s disease Compared with the urine 8 OHdG assay that mainly reflects the balance between oxidative damage and the repair rate of the whole body directly quantifying the 8 OHdG content in different cells tissues in normal and disease states would allow tissue specific oxidative damage of DNA to be identified Therefore more
9. g the solution already fluorescent in the wells with PBS at 1 5 or 1 10 ratio The RFU ratio remains unchanged in the designated assay wells 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 23 P 6004 DNA sample is not properly Ensure the DNA sample is in good extracted or purified quality 260 280 ratio should be gt 1 7 with no or minimal RNA contamination No signal or weak Sample amount added into the Ensure a sufficient amount of DNA is signal only in sample wells is insufficient used as indicated in Step 3 oe Little or no 8 OHdG contained in N A the sample Uneven fluorescent Delayed fluorescence Ensure fluorescence development development development in the wells solution is added sequentially and is consistent with the order you added the other reagents e g from well A to well G or from well 1 to well 12 If using 12 or more wells a multi channel pipette should be used so that the addition of fluorescence development solution can be completed as quickly as possible lt 20 seconds 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 Iso
10. have 100 of 8 OHdG SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store NC PC DA ES and FD at 20 C away from light 2 Store WB CA DS FE and 8 Well Assay Strips at 4 C away from light 3 Store remaining components BS and DB at room temperature away from light Note Check if wash buffer WB contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved All components of the kit are stable for 6 months from the date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette Aerosol resistant pipette tips Microplate reader capable of reading fluorescence 530ex 590em 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Oood a0 Plate seal or Parafilm M 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 09 23 P 6004 O Distilled water O 1X TE buffer pH 7 5 to 8 0 O Isolated DNA of interest GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric is tested against predetermined specifications to ensur
11. ing 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 PC at a concentration of 100 pg ul as prepared in Step 2f For the standard curve add 1 ul of Diluted PC at concentrations of 5 to 200 pg ul see the chart in Step 2 The final amounts should be 5 10 20 50 100 and 200 pg per well 2 For optimal binding sample DNA volume added should not exceed 8 ul Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min Remove the BS Binding Solution from each well Wash each well with 150 ul of the Diluted WB 1X Wash Buffer each time for three times 4 8 OHdG DNA Capture Add 50 ul of the Diluted CA to each well then cover and incubate at room temperature for 60 min Remove the Diluted CA solution from each well Wash each well with 150 ul of the Diluted WB each time for three times Add 50 ul of the Diluted DA to each well then cover and incubate at room temperature for 30 min Remove the Diluted DA solution from each well Wash each well with 150 ul of the Diluted WB each time for four times Add 50 ul of the Diluted ES to each well then cover and incubate at room temperature for 30 min Remove the Diluted ES solution from each well Wash each well with 150 ul of the Diluted WB each time for five times 5 Signal Detection a Add 50 ul of Fluorescence Development Solution to each well and in
12. ip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A NC NC Sample Sample Sample Sample B PC PC 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 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 Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A NC NC Sample Sample Sample Sample B PC 5 pg ul PC 5pg ul Sample Sample Sample Sample c PC10 pg ul PC10 pg ul Sample Sample Sample Sample D PC 20 pg ul PC 20 pg ul Sample Sample Sample Sample E PC 50 pg ul PC 50 pg ul Sample Sample Sample Sample F PC 100 pg ul PC 100 pg ul Sample Sample Sample Sample G PC 200 pg ul PC 200 pg ul Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 3 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1 well 8 wells 16 wells 48 wells 96 wells 1 strip 2 strips 6 strips 12 strips Diluted WB 2 5 ml 20 ml 40
13. ive 8 OHdG 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 Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 6004 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 6004 48 Cat P 6004 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 47 BS Binding Solution 5 ml 10 ml RT NC Negative Control 10 ug ml 10 ul 20 ul 20 C PC Positive Control 8 OHdG 1 ug ml 10 ul 20 ul 20 C CA Capture Antibody 100 X 28 ul 55 ul 4 DA Detection Antibody 1000 X 6 ul 12 ul 20 C ES Enhancer Solution 5 ul 10 ul 20 C FD Fluoro Developer 10 pl 20 ul 20 C FE Fluoro Enhancer 10 ul 20 ul 47 DB Dilution Buffer 4ml 8 ml RT 8 Well Assay Strips With Frame 6 12 47 User Guide 1 1 RT Spin the solution down to the bottom prior to use Note The NC Negative Control is an oligos containing no 8 OHdG The PC Positive Control is an 8 OHdG oligos and normalized to
14. lation 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 Methylated and Hydroxymethylated DNA Quantification P 1034 MethylFlash Methylated DNA Quantification Kit Colorimetric P 1035 MethylFlash Methylated DNA Quantification Kit Fluorometric P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric P 1037 MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric Hydroxymethylated DNA Immunoprecipitation P 1038 MethylFlash Hydroxymethylated DNA Immunoprecipitation hmeDIP Kit DNA Damage and Repair OP 0001 EpiQuik Superoxide Dismutase Activity Inhibition Assay Kit Colorimetric OP 0004 CytoX Red Cell Proliferation Cytotoxicity Assay Kit OP 0005 CytoX Violet Cell Proliferation Cytotoxicity Assay Kit P 6003 EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Colorimetric P 6001 EpiQuik In Situ DNA Damage Assay Kit DNA Damage and Repair Antibodies See http Awww epigentek com catalog dna damage repair c 35_70 html 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 23 Epigentek Group Inc All rights reserved Products are for research use only P 6004
15. ml 120 ml 240 ml BS 80 ul 640 ul 1300 ul 3900 ul 8000 ul Diluted CA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted ES 50 ul 400 ul 800 ul 2400 ul 4800 ul DS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml SS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml NC N A 0 5 ul 1l 0 5ul 2yul iul 4u J2ul 8ul PC N A 0 5 ul 1l 0 5 ul 2yul iul 4u J2ul 8ul 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 23 P 6004 TROUBLESHOOTING Problem Possible Cause Suggestion No signal in both the positive control and sample wells 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 prior to adding the positive control and sample The bottom of the well is not completely covered by the BS 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
16. ommend using Epigentek s series of DNA isolation kits 10000 9000 8000 Bind DNA to assay well 7000 6000 gt Wash wells then add W 5000 capture antibody cc 4000 3000 Wash wells then add detection antibody and 2000 enhancer solution l 1000 0 T T T T 1 Add fluoro developing 0 50 100 150 200 250 solution for fluorescence then measure RFU 8 OHdG control pg Schematic procedure of the EpiQuik 8 OHdG 8 OHdG standard control was added into the assay wells at DNA Damage Quantification Direct Kit different concentrations and then measured with the Fiance Quantific angn Direct KI EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment 1 Starting Materials Input DNA Amount DNA amount can range from 100 ng to 300 ng per reaction An optimal amount is 300 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 see Related Products section DNA Storage Isolated genomic DNA can be stored at 4 C short term or 20 C long term until use 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 Web www epigentek com Printed 2014 0
17. sufficient amount of positive control gt 50 pg and sample 300 ng is added into the wells Incorrect fluorescence reading Check if the appropriate fluorescence filters 530ex 590em are used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly secured after each opening or use No signal or weak signal in only the positive control wells The positive control is insufficiently added to the well in Step 3c Ensure a sufficient amount of positive control DNA is added The PC Positive Control is degraded due to improper storage conditions Follow the Shipping amp Storage guidelines of this User Guide for storage of PC Positive Control High background present in the negative control wells or RFU in some of standard and sample wells is out of the fluorescent reading range Insufficient washing of wells Check if washing recommendations at each step are performed according to the protocol Contaminated by sample or positive control Ensure the well is not contaminated by the sample or positive control DNA or from the use of contaminated tips Incubation time is too long The incubation time should not exceed 2 h at Step 3d and not exceed 45 min at step 4d Over development of fluorescence Decrease the development time in Step 4a Also RFU can be re measured after dilutin
18. tes interference from high molecular weight compounds such as carbohydrates and proteins that are often seen in competitive 8 OHdG assays e Detection accuracy is highly correlated with and close to HPLC or LC MS analysis e Highly convenient assay with direct use of DNA isolated from cells or tissues no need for DNA digestion or hydrolysis e Universal positive and negative controls are included which are suitable for quantifying 8 OHdG from any species e Strip well microplate format makes the assay flexible for manual or high throughput analysis e Simple reliable and consistent assay conditions 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 23 Epigentek Group Inc All rights reserved Products are for research use only P 6004 PRINCIPLE amp PROCEDURE The EpiQuik 8 OHdG DNA Damage Quantification Direct Kit Fluorometric contains all reagents necessary for the quantification of Oxidative DNA damage 8 OHdG In this assay DNA is bound to strip wells that are specifically treated to have a high DNA affinity 8 OHdG is detected using capture and detection antibodies The detected signal is enhanced and then quantified fluorometrically by reading the fluorescence in a fluorescence microplate reader The amount of 8 OHdG is proportional to the fluorescence intensity measured Prepare genomic DNA We rec
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