CpGenome Universal Methylated DNA

PCR Methylation specific PCR - Mammalian DNA

Experiment
PCR Methylation specific PCR - Mammalian DNA
Product
CpGenome Universal Methylated DNA from Merck Millipore
Manufacturer
Merck Millipore

Protocol tips

Upstream tips
Aliquote in small quantity to avoid freeze-thaw cycles and Storage at -70°C for long-term use (2-3 yrs)

Publication protocol

Quantitative and Allelic Methylation Analyses by qMSP Followed by Pyrosequencing
Quantitative and allelic methylation analyses were performed using qMSP and melting analyses followed by pyrosequencing of methylation-positive samples being heterozygous for the rs16906252 SNP (). The genomic location of the primers is shown in . For normalization purposes, we used an assay based on Alu sequences depleted of CpG sites by evolutionary deamination (). This assay is less susceptible to normalization errors caused by aneuploidy and copy number changes often observed in cancer cells. Additional details are in the .

Quantification of Methylation Levels Using qMSP
Methylation levels of the samples were estimated relative to universal methylated DNA (Chemicon, Millipore, Billerica, MA) using the 2 quantification approach (), in which ΔΔCt = glioblastoma sample (Ct – Ct) − universal methylated DNA (Ct − Ct) (). This approach implies that the assays have approximately the same PCR efficiency () (). For the assay, = 2.0; for the Alu assay, = 1.9. Samples were analyzed in duplicate and the average Ct values were used in the calculations. Only samples for which both replicates amplified in the assay having a melting profile resembling the melting profile of universal methylated DNA were considered methylation positive. Samples with a mean Ct value above 17 in the Alu assay were bisulfite converted again and repeated. Samples with methylation levels below 0.1% were analyzed as unmethylated. This technical cutoff was defined following an evaluation of a serial dilution series of methylated DNA into unmethylated. Samples with a methylation level below 5% were scored as low-level methylation; samples with a methylation level between 5% and 20% as medium; and samples with a methylation level above 20% as high. No template control and unmethylated DNA, prepared as described (), were included as negative controls.

Standard Bisulfite Pyrosequencing
PCR and pyrosequencing were performed using the Therascreen (R) MGMT Pyro (R) kit according to the manufacturer’s instructions with slight modifications (). The CpG sites analyzed by the standard pyrosequencing assay are shown in . Samples with a mean methylation level below 10% were considered unmethylated. Samples with a mean methylation level between 10% and 25% were scored as low-level methylation; samples with a mean methylation level between 25% and 50% as medium; and samples with a mean methylation level above 50% as high.

Full paper   Login or join for free to view the full paper.

Reviews

CpGenome Universal Methylated DNA from Merck Millipore has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Discussion

4 years ago

Author: United Kingdom

How “strong” is the PCR product of methylation specific PCR?

Does anyone know how “strong” the PCR product of methylation specific PCR is? I kept my PCR products at 4C for about 3 weeks and then at room temperature for another week. Will I be able to use them for sequencing?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Methylation specific PCR - Mammalian DNA using CpGenome Universal Methylated DNA from Merck Millipore.

Paper title
Assessment of Quantitative and Allelic Methylation Patterns as a Prognostic Marker in Glioblastoma
Full paper
Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Merck Millipore for CpGenome Universal Methylated DNA below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Methylation specific PCR - Mammalian DNA using CpGenome Universal Methylated DNA from Merck Millipore. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms