About OneStep qMethyl™
The OneStep qMethyl™ Kit from Zymo Research provides a simple, straightforward, and bisulfite-free procedure for rapid, locus-specific DNA methylation assessment. Simply add DNA into the appropriate reaction mix then quantitate via real-time PCR OneStep!
|Format||Provided Test Reaction PreMix and Reference Reaction PreMix contain SYTO®9 dye. Kit can be purchased without dye in the premixes (see OneStep qMethyl™ -Lite)|
|Processing Time||4.5 hrs|
|Input||High quality DNA suitable for use in restriction enzyme digestion. Require A260/280 > 1.8, A260/230 > 1.8.|
|Storage||Store at -20°C for up to 12 months. Avoid repeated freeze/thawing of reagents. Prolong storage at -80°C. 2X Test Reaction PreMix and 2X Reference Reaction PreMix are light sensitive, minimize light exposure.|
Simple, One Step DNA Methylation Determination
Unlike conventional qAMP procedures that contain multiple steps, the OneStep qMethyl™ method integrates the workflow into a simple, single step reaction. This minimizes errors that can occur during setup and the likelihood of contamination while allowing for rapid and accurate DNA methylation level detection.
The OneStep qMethyl™ Kit is used for the detection of locus-specific DNA methylation via the selective amplification of a methylated region of DNA. This is accomplished by splitting any DNA to be tested into two parts: a "Test Reaction" and a "Reference Reaction" (see Figure 1 below). DNA in the Test Reaction is digested with Methylation Sensitive Restriction Enzymes (MSREs) while DNA in the Reference Reaction is not. The DNA from both samples is then amplified using real-time PCR in the presence of SYTO®9 fluorescent dye and then quantitated.
Schematics A and B (above) illustrate the sample workflow of Non-methylated DNA and Methylated DNAs. In both cases the DNA is divided in two parts; a Test Reaction and a Reference Reaction. Test Reaction samples are MSRE digested while the Reference Reaction samples are not (mock digested). Following digestion, DNA from both samples is used for real-time PCR. The white lollipops in the image represent unmethylated cytosines and black lollipops methylated cytosines in CpG dinucleotide context. Following real-time PCR, amplification plots (C and D) demonstrate non-methylated DNA exhibits large differences in the Ct values for Test and Reference Reactions (C) while highly methylated DNA samples exhibit little difference (D).
The following protocol illustrates the use of the OneStep qMethyl™ procedure for DNA methylation detection at a single region. This allows for the processing of up to 22 DNA samples (in duplicate) using a 96-well real-time PCR plate. The provided Human Methylated & Non-methylated DNA Standards should be processed along with the samples for the purpose of validating the combined MSRE digestion/real-time PCR step (see Appendix III, page 10). The following should serve as a guideline when setting up your own experiment. The format is also compatible with thin-walled PCR tubes and tube strips.
I. Preparation of Test Reaction and Reference Reaction Mixtures4.
Protocol for Methylation Detection Levels for Multiple DNA Samples
Test Reaction (per well)
10 µl 2X Test Reaction PreMix (contains MSREs)
Reference Reaction (per well)
10 µl 2X Reference Reaction PreMix
Make a Test and Reference Reaction master mix to accommodate the 22 DNA samples and the Methylated & Non-methylated DNA Standards. Add 15 µl of Test and Reference Reaction master mix to the wells of a 96-well real-time PCR plate (not provided).
Add 5 µl (20 ng) of the appropriate DNA sample to those designated wells (in duplicate).
II. OneStep MSRE Digestion/Real-Time PCR
Reaction conditions for the combined MSRE Digestion/Real-Time PCR processes have been optimized for direct input, digestion, and real-time amplification of DNA samples. However, annealing temperature and elongation time may need to be optimized depending on the design of the primers. The parameters below are for the supplied MGMT primers and should be used as a guideline when setting up your own experiment. Typically, between 40-45 cycles is recommended for the amplification of most DNA templates.
Note: a setting that is compatible with SYBRgreen® should be compatible with the SYTO 9® dye in this kit.
Perform real-time PCR using the following parameters.
* If necessary, the MSRE Digestion can be performed at 37oC using an incubator or similar device.
**The annealing temperature and extension time with may vary with user designed primers and the size of the amplicon. Therefore, it may be necessary to adjust and optimize these conditions.
III. Data Analysis
The methylation level for any amplified region can be determined using the following equation:
Percent Methylation = 100 x 2-ΔCt where ΔCt = the average Ct value from the Test Reaction minus the average Ct values from the Reference Reaction
The table (below) represents actual real-time PCR data from the OneStep qMethyl™ procedure using the Human Non-methylated DNA Standard and MGMT primers (performed in duplicate).
To determine the methylation level of the Human Non-methylated DNA standard,
1. Calculate the average Ct values for Test and Reference Reactions.
2. Determine the ΔCt by subtracting the average Ct value of Reference Reaction from the average Ct value of Test Reaction.
ΔCt = 33.82 - 29.79 = 4.03
3. Substitute the ΔCt value into the equation: 100 x 2-ΔCt
100 x 2-4.03 = 6%
Using this equation, the methylation level of the Human Non-methylated DNA Standard is determined to be 6 % at the region spanned by the MGMT primers. The actual value is ~5% as determined by bisulfite sequencing methods. Due to the low background level of methylation in Zymo’s non-methylated standard cell line, the results of the calculation are within the expected limit of methylation detection.
Accurate DNA methylation detection using the OneStep qMethyl Kit was used to assess the percent of DNA methylation in the promoter region of two potential epigenetic biomarkers for pregnancy-related diseases. The DNA methylation detection was able to correlate the increase in DNA methylation with the increase in cellular invasion using DNA samples from first trimester placentas that were known to have an outgrowth potential.