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FAQs : EpiTect Methyl II PCR System

Real-time PCR and PCR Array Technical FAQ

The EpiTect Methyl II PCR System use the MethylScreen™ Technology provided under license from Orion Genomics, LLC.

Q: How can I be sure that the restriction enzyme digestion is complete?
A: The protocol uses an excess of each restriction enzyme and a sufficient incubation time relative to the recommended DNA amounts. This combination is more than sufficient to completely digest the DNA sample. The larger the difference between the Ct values of the mock and double digests [W = Ct (Mo) – Ct (Msd)], the more complete the restriction enzyme digestion. Because Ct values are inversely and exponentially related to the initial amount of DNA material, each unit of cycle difference between these digests represents an additional two-fold difference in the amount of DNA. For example, a five-cycle difference means that the double digest contains only (100 x 2 ^ -Ct = 100 x 2 ^ 5 = ) 3.125 percent of the DNA of the mock digest, meaning that the reaction is therefore (100 - 3.125 = ) 96.875 percent complete.

Q: Can this technology reliably characterize heterogenous tissue samples?
A: Thanks to the sensitive and quantitative nature of real-time PCR, hypermethylated DNA can be detected even when the relevant cells are present at only five percent of a total population containing mostly normal stromal cells. This level of sensitivity equals bisulfite-sequencing and bisulfite-PCR methods. In contrast, Methylation-Specific PCR (MSP) detects sequences only when the normally inefficient bisulfite conversion is 100 percent successful, thus having the decreased sensitivity.

Q: Are EpiTect Methyl II PCR Arrays or Assays applicable to FFPE samples?
A: No. Under the current protocol, FFPE samples are not suitable for analysis with EpiTect Methyl II PCR Arrays and Assays. This is because genomic DNA from FFPE samples is single-stranded due to the heating step involved in the DNA isolation process, making it undigestible by either Methylation-sensitive or Methylation-dependent enzymes. Please refer to the User Manual of your chosen genomic DNA isolation kit. If there is a 95 ºC heating step involved, please do not use the kit to isolate genomic DNA for EpiTect Methyl II PCR Arrays or Assays. Instead, use the recommended genomic DNA isolation Kit in the User Manual of EpiTect Methyl II PCR Arrays or Assays.

For analyzing methylation status of DNA isolated from FFPE tissues, we recommend using EpiTect Plus FFPE Bisulfite Kits for one-step isolation and bisulfite conversion of the DNA, followed by any detection method optimized for use with FFPE samples (e.g., Pyrosequencing).

Q: Why is my EpiTect Methyl II PCR assay "failed" as indicated in the QC page of the data analysis Excel file?
A: That is because the Ct difference between mock digestion and double digestion is less than 2, which means that less than 75% of the input genomic DNA is resistant to the digestion. Therefore, the result from such digestions is unreliable in DNA methylation analysis. So we labeled such an assay as failure and exclude it from data analysis.

Q: How are the primers in the DNA methylation PCR Array designed?
A: The primers are designed around CpG islands known to be hypermethylated under relevant biological conditions or in relevant biological samples. Besides the usual specific requirements that real-time PCR primers must meet, the amplicon sequence must contain both restriction sites for both the methylation-sensitive and methylation-dependent enzymes. As a result, the amplicon lengths are longer than those seen for RT-PCR, which are around 150 to 400 bp. Our design algorithm also accounts for the GC-rich nature of the genomic DNA sequences that tend to make primer design more difficult, especially in and around CpG islands. Each primer pair is also experimentally verified at the bench for a single peak in the dissociation curve, and high amplification efficiencies

Q: Do I need an internal methylated DNA reference?
A: No. For any given target sequence, the same primer pair is used to amplify the four different digests allowing the PCR results to be reliably and directly compared. In contrast, bisulfite conversion-based PCR methods use two different pairs of primers to amplify either methylated or unmethylated templates of a given target sequence after conversion. Differences in their amplification efficiencies could cause biased results, requiring normalization to in vitro methylated reference DNA. However, since the primers need similar amplification efficiencies and the methylation need to be consistently complete, reliable results can only be achieved after careful trial and error based optimization and additional cost as well.

Q: Does this method detect methylation at specific CpG dinucleotides?
A: No. This method examines the methylation status across a CpG-rich sequence. It is very convenient when quickly screening for changes in methylation status as well as for detecting new biomarkers. For confirming results obtained with the EpiTect Methyl II PCR system, and for determining the exact methylation levels of specific CpG sites, we recommend using a quantitative, sequence-based method such as bisulfite Pyrosequencing.

Q: Can I reliably detect intermediately methylated DNA with this technology?
A: Yes. The total, hypermethylated (> 60 percent methylated), and unmethylated (0 percent methylated) amounts of DNA in the sample are each directly and reliably detected. The total amount of DNA minus the hypermethylated and unmethylated DNA amounts yields the amount of intermediately methylated DNA (between 0 and 60 percent methylated). Only Pyrosequencing and bisulfite Sanger sequencing have this same capability. Lower extents of hypermethylation have increasingly been correlated with biological phenotypes when coincident with larger extents of intermediate methylation.

Q: What is the minimum amount of genomic DNA required for EpiTect Methyl II PCR analysis?
A: For digestion, we recommend using 0.5-4ug (0.125-1ug per digestion) of genomic DNA for EpiTect Methyl II PCR Arrays and 0.25-0.5ug (0.0625-0.125ug per digestion) for single qPCR assays. However, you should not use less than 0.25ug (0.0625ug per digestion) of genomic DNA for digestion. For PCR reactions, we recommend using 5-10ng per PCR reaction and at least 2ng genomic DNA should be included in each PCR reaction.

Q: What does a typical EpiTect Methyl II PCR Assay or Array include?
A: A typical order for EpiTect Methyl II PCR Arrays or Assays includes the EpiTect Methyl DNA Restriction Kit, EpiTect Methyl II PCR Assays or Arrays, and instrument specific SYBR Green Master Mix.

Q: Do EpiTect Methyl II PCR Arrays or Assays need a specific PCR instrument to run?
A: No. The EpiTect Methyl II PCR Arrays or Assays can be run on any real-time PCR instrument on the market. You just need to purchase the instrument specific SYBR Green Master Mix accordingly from SABiosciences.

Q: Will pipetting error affect the EpiTect Methyl II PCR Array results?
A: The passive reference dyes in the PCR SYBR Green master mixes, such as ROX and Fluorescein, are used by the real-time PCR instruments to normalize variation from well to well. Therefore, these systems tolerate volume variations caused by pipetting error or evaporation. Even a 20% pipetting error causes only 0.05-cycle differences in Ct values.

Q: How can I prevent the evaporation of reaction volume from the wells?
A: Be sure to carefully and completely seal the PCR Array with the optical thin-wall 8-cap strips or the optical adhesive film before placing it into your thermal cycler.

Q: Why can't I find the DNA methylation qPCR Assay for my gene of interest?
A: There are three possible reasons. First, there is no predicted CpG island in the promoter region (defined as 5 kb upstream to 3 kb downstream of the transcription start site) of your gene of interest. Second, part of our primer design criteria is that the amplicon should include both methylation sensitive and dependent enzyme cutting sites. If your CpG island of interest does not contain these, we are unable to design primers for it. Third, the gene symbol you input may not be correct. You will be informed of those cases in the search result page.

Q: Can you custom design the primers for the CpG island outside of the defined promoter region?
A: Yes, we can under the condition that you provide all the following information to us: 1) the location of CpG island of your interest; 2) the sequence of the CpG island; 3) the specific region that you are interested in. Once we receive all the required information from you, our bioinformatics group will try to design the primers. However, for rare cases, the primers may not be available due to the failure of fulfillment of our stringent primer design criteria.

Q: Can I use the default PCR program to run EpiTect Methyl II PCR Arrays or Assays?
A: Absolutely not. Due to the GC-rich nature of CpG islands, EpiTect Methyl II PCR Primers require a very specific combination of PCR temperature cycle and chemistry to obtain reliable and accurate DNA methylation analysis. Without the correct PCR cycling program, your DNA methylation analysis will fail due to the non-specific amplification during PCR. Do double check the manual for the appropriate PCR cycle for EpiTect Methyl qPCR DNA methylation analysis for you instrument model.

Q: Are the methylation sensitive restriction enzymes also hemimethylation sensitive?
A: Yes, the methylation sensitive restriction enzymes are hemi-methylation sensitive.

Q: Are the methylation dependent restriction enzymes also hemimethylation dependent (i.e. will they cut hemi-methylated DNA)?
A: Yes, the methylation-dependent restriction enzymes are hemimethylation dependent. They will cut hemi-methylated DNA.