SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric)
Uses
The SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) is suitable for detecting global DNA hydroxymethylation levels using DNA isolated from any species including 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. Both single stranded DNA and double stranded DNA with a size of 200 bps to full length are suitable for use.
Input DNA
The amount of DNA for each assay can be 50 to 200 ng. For the most ideal quantification, the input DNA amount should be 100 ng, as hydroxymethylated DNA varies from tissue to tissue and can be less than 0.6% of total DNA in some species.
Internal Control
Both negative and positive DNA controls are provided in this kit. A standard curve can be performed (range: 0.02% to 0.8%). Because global hydroxymethylation can vary from tissue to tissue, and from normal and diseased states, 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 hydroxymethylated 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.
Kit Contents
| Component | 96 Reactions Cat. #P-7101-96 | Storage Upon Receipt |
|---|---|---|
| WB (10X Wash Buffer) | 14 ml | 4°C |
| HBS (High Binding Solution) | 10 ml | RT |
| NC (Negative Control containing 0% 5-hmC, 50 ng/µl)* | 100 µl | -20°C |
| PC (Positive Control containing 2% 5-hmC, 50 ng/µl)* | 20 µl | -20°C |
| hmcAb (5-hmC Antibody, 1000X)* | 10 µl | 4°C |
| MSI (Signal Indicator, 1000X)* | 10 µl | -20°C |
| ES (Enhancer Solution, 1000X)* | 10 µl | -20°C |
| DS (Developer Solution) | 10 ml | 4°C |
| SS (Stop Solution) | 10 ml | RT |
| 8-Well Assay Strips (With Frame) | 12 | 4°C |
* Spin the solution down to the bottom prior to use.
The NC is unmethylated DNA containing 0% of 5-hydroxymethylcytosine. The PC is hydroxymethylated DNA containing 2% of 5-hydroxymethylcytosine.
Shipping & Storage
The kit is shipped in two parts: the first part at ambient room temperature and the second part on frozen ice packs to maintain approximately 4°C.
Upon receipt: (1) Store NC, PC, MSI and ES at -20°C away from light; (2) store WB, hmcAb, DS, and 8-Well Assay Strips at 4°C away from light; (3) Store HBS and SS at room temperature away from light.
All components of the kit are stable for 6 months from the date of shipment, when stored properly.
Check if WB 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
Principle & Procedure
The SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) contains all reagents necessary for the quantification of global DNA hydroxymethylation. In this assay, DNA is bound to strip-wells with a high binding buffer. The hydroxymethylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of hydroxymethylated DNA is proportional to the OD intensity measured.
Starting Materials
For the best results, please read the protocol in its entirety prior to starting your experiment.
Input DNA Quality and Amount: Input DNA should be relatively pure with 260/280 ratio >1.6 and can be diluted with water or TE buffer. The DNA amount can range from 50 ng to 200 ng per reaction. However, we recommend using 100 ng of DNA, which is the optimized input amount for the best results.
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. Working Buffer and Solution Preparation
Prepare Diluted WB (1X Wash Buffer) by adding 12 ml of WB (10X Wash Buffer) to 108 ml of distilled water.
This Diluted WB can now be stored at 4°C for up to six months.
The anticipated approximate volumes of reagents needed are reflected below for this assay.
| Reagents | 1 well | 8 wells (1 strip) | 16 wells (2 strips) | 48 wells (6 strips) | 96 wells (12 strips) |
|---|---|---|---|---|---|
| Diluted WB | 1 ml | 8 ml | 16 ml | 48 ml | 96 ml |
| HBS | 100 µl | 800 µl | 1600 µl | 4800 µl | 9600 µl |
| 5-hmC Detection Complex | 50 µl | 400 µl | 800 µl | 2400 µl | 4800 µl |
| 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 | 2 µl | 2 µl | 60 µl | 60-100 µl |
| PC | N/A | N/A | Optional | 6 µl | 6-12 µl |
2. Standard or Positive Control (PC) Curve Preparation
Dilute 6 µl of PC with 9 µl of NC to make Diluted PC (0.8%). Mix well. Then, prepare 6 concentration points for the control by combining 0.8% PC and NC according to the following chart. Mix well to ensure the accuracy of the concentration.
| Tube | 5-hmC PC (0.8%) | NC | Resulting PC Concentration |
|---|---|---|---|
| 1 | 0.5 µl | 19.5 µl | 0.02%, 0.01 ng in 50 ng/µl DNA |
| 2 | 0.5 µl | 9.0 µl | 0.04%, 0.02 ng in 50 ng/µl DNA |
| 3 | 1.0 µl | 9.0 µl | 0.08%, 0.04 ng in 50 ng/µl DNA |
| 4 | 1.0 µl | 3.0 µl | 0.2%, 0.1 ng in 50 ng/µl DNA |
| 5 | 2.0 µl | 2.0 µl | 0.4%, 0.2 ng in 50 ng/µl DNA |
| 6 | 4.0 µl | 0.0 µl | 0.8%, 0.4 ng in 50 ng/µl DNA |
1) The above volumes will be sufficient for one standard curve in duplicate (12 wells total). The PC concentrations are based on per assay well, not per microliter; 2) Keep each of 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 Strip Well Setup
Review the configuration of the strip-well plate setup for standard curve preparation strips, showing strip 1 to 6 (7 through 12 can be configured as Sample). The controls and samples can be measured in duplicate, loaded vertically instead of horizontally.
| Well # | Strip 1 | Strip 2 | Strip 3 | Strip 4 | Strip 5 | Strip 6 |
|---|---|---|---|---|---|---|
| A | NC | PC 0.2%/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| B | NC | PC 0.2%/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| C | PC 0.02%/well | PC 0.4%/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| D | PC 0.02%/well | PC 0.4%/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| E | PC 0.04%/well | PC 0.8%/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| F | PC 0.04%/well | PC 0.8%/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| G | PC 0.08%/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
| H | PC 0.08%/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
4. DNA Binding
(1) To reduce cross variation between replicates, it is important to load the wells in vertical formation according to the plate layout depicted above. (2) For the positive controls, total DNA is 100 ng per well with different hydroxymethylation percentages (0.02%, 0.04%, 0.08%, 0.2%, 0.4% and 0.8%). (3) For optimal binding and to reduce pipetting error, sample DNA volume added should be 2 µl or more, but should not exceed 5 µl. (4) To ensure that NC, PC, and sample DNA are completely added into the wells, the DNA should be mixed well before use and the pipette tip should be placed into the HBS solution in the well and aspirated in/out 1-2 times. Changing the tips each time when adding the sample will increase sample volume accuracy added into each well
5. 5-hmC Level Detection and Signal Measurement
The color development time may vary from 1-10 minutes based on the speed of color change, but is typically 3-4 minutes.
6. 5-hmC% Calculation
To calculate percentage of hydroxymethylated DNA, first generate a standard curve and plot the OD values versus the PC at each percentage point. Next, determine the slope (OD/1%) of the standard curve using linear regression (Microsoft Excel can be used) and the most linear part (at least 4 concentration points including NC point) of the standard curve for optimal slope calculation. Now, calculate the percentage of hydroxymethylated DNA (5-hmC) in total DNA using the following formula:
S is the amount of input sample DNA in ng.
In the event that the standard curve is flat due to high ODs starting from the lowest %PC or is flat at high %PCs because of a saturated signal intensity due to extended color development time, the 5-hmC% can be calculated with logarithmic or polynomial second order regression (see the Appendix section).
A Brief Overview
5-hydroxymethylcytosine (5-hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5-mC, a reaction mediated by the ten-eleven translocation (TET) family of 5-mC-hydroxylases.
5-hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5-hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5-hmC content (DNA hypo-hydroxymethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well.
Accurate and convenient detection of 5-hmC would be extremely useful for identifying and understanding the global hydroxymethylation changes that occur in DNA during various physiological and pathological processes such as in cancer. To address this, EpigenTek offers a series of Hydroxymethylated DNA Quantification Kits to quantify 5-hmC or hydroxymethylated DNA and further refines its hydroxymethylated DNA assay expertise with the development of the SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric). This kit has the following advantages and features:
- Fast - Reduced steps so that the entire procedure only needs 1 hour*
- Robust - Improved kit composition allows the assay to have a greater "signal window" with reduced variation between replicates
- Convenient - Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps
- Sensitive - Detection limit can be as low as 0.01% hydroxymethylated DNA from 100 ng of input DNA
- Specific - High specificity to 5-hmC, with no cross-reactivity to unmethylated cytosine or methylated cytosine within the indicated concentration range of the sample DNA
- Universal - Positive and negative controls allow detection of DNA hydroxymethylation in any species from either single-stranded or double-stranded input DNA
- Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis
* Based on a single sample assay in duplicate
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 HBS (High Binding Solution). | Ensure the solution coats the bottom of the well by gently tilting from side to side or gently shaking the plate several times. | |
| Incubation time and temperature are incorrect. | Ensure the incubation time and temperature described in the protocol are followed correctly. | |
| Insufficient input materials. | Ensure that a sufficient amount of positive control and samples are added into the wells. | |
| Incorrect absorbance reading. | Check if appropriate absorbance wavelength (450 nm) is used. | |
| Kit was not stored or handled properly. | Ensure all components of the kit were stored at the appropriate temperature and the caps are tightly capped after each opening or use. | |
| No signal or weak signal in only the positive control wells | The PC (Positive Control) DNA is insufficiently added to the well in Step 4d. | Ensure a sufficient amount of positive control DNA is added. |
| The PC (Positive Control) is degraded due to improper storage conditions. | Follow the Shipping & Storage guidance in this User Guide for storage of PC (Positive Control). | |
| High background present in the negative control wells | Insufficient washing of wells. | Check if washing recommendations at each step are 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. | |
| Over development of color. | Decrease the development time in Step 5d before adding SS (Stop Solution) in Step 5e. | |
| Large variation between replicate wells | Horizontal positioning of well replicates causes inconsistent delays in pipetting and loading of reagents. | Follow the vertical layout example provided in Step 3. Ensure loading of reagents is also in vertical order with a multi-channel pipette, especially when adding DS (Developer Solution) and SS (Stop Solution) in Step 5. |
| Color reaction is not evenly stopped due to an inconsistency in pipetting time or in pipetting volume. | Ensure DS (Developer Solution) and SS (Stop Solution) is added at the same time between replicates or otherwise maintains a consistent timing in between each addition of solutions. 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 µl) are completely added into the wells. Pay special attention at the steps of adding DNA samples and preparing 5-hmC Detection Complex Solution. | |
| Color reaction is not evenly stopped due to an inconsistent order of adding solutions. | Ensure all solutions, particularly DS (Developer Solution) and SS (Stop Solution), are added in the same order each time as all other solutions. | |
| Residue wash buffer is present in some of the wells. | Ensure the wash buffer is completely removed at each wash step. | |
| Splashing of reagents between wells. | Pipette carefully against the sides of the wells to avoid splashing. | |
| Temperature variations across the plate. | Ensure plates are evenly and fully covered during incubation steps in a stable temperature environment, away from drafts. | |
| Large variation between sample replicate wells only | Sample DNA is sedimented or uneven prior to loading to wells. | Mix your sample DNA sufficiently and evenly prior to loading it into wells. |
| hmcAb (5-hmC Antibody) vial appears to 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 hmcAb vial until you restore the correct, intended volume according to the Kit Contents described in this User Guide. Mix and centrifuge prior to use. |
Appendix
Method 1: Target Calculation Using Logarithmic Regression
Use this method when the standard curve is flat due to high ODs starting from the lowest standard concentration.
Here, X = target level represented on the standard curve X-axis; Y = Sample OD; a is the regression coefficient and b is the Y-intercept.
*Microsoft Excel's logarithmic regression function can be used for easy and convenient calculation.
Here, S is the amount of input sample in ng.
Example Calculation
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel. In the figure above, a = 0.2508, b = 1.1329, Y = 1.274. Input sample amount was 100 ng.
Target percentage of the sample was calculated accordingly:
Method 2: Target Calculation Using Polynomial Second Order Regression
Use this method when the standard curve is flat due to a saturated signal intensity at high standard concentrations.
Here, X = target level represented on the standard curve X-axis; Y = (sample OD value - negative control or blank OD) represented on the standard curve Y-axis; a and b are regression coefficients.
*Microsoft Excel's polynomial regression function can be used for easy and convenient calculation.
Here, S is the amount of input sample in ng.
Example Calculation
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel. In the figure above, a = -0.0865, b = 0.6373, Y = (0.621 - 0.060) = 0.561. Input sample amount was 100 ng.
Target percentage of the sample was calculated accordingly:
If the background-corrected sample OD is less than zero or falls outside the measurable range of the standard curve, the target level should not be calculated by extrapolation. The sample should be considered below the quantifiable range or re-assayed at an appropriate dilution, as applicable.
General Product Information
Quality Control
Each lot of the SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) 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. Be sure to use the latest User Guide for this kit which can be accessed online at www.epigentek.com/datasheet.
Usage Limitation
The SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) is for research use only and is not intended for diagnostic or therapeutic applications.
Intellectual Property
The SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) and methods of use contain proprietary technologies by EpigenTek.