DNA Methylation

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17 What is the best protocol to treat culture with 5-aza-2'-deoxycytidine to block methylation?
By Priyanka Bhosale · Advanced Centre for Treatment, Research and Education in Cancer

Jaydeep Bhat · Universitätsklinikum Schleswig - Holstein

Yea! Stability is the biggest issue with 5-Aza drugs. I had experienced this very well. However, one should not used this drug for more than 1-2 weeks... [more]

Yea! Stability is the biggest issue with 5-Aza drugs. I had experienced this very well. However, one should not used this drug for more than 1-2 weeks, when dossolved in medium. In my experiments, I always prefer to use freshly (around 1-1.5 week), then I don't have any stability issue. Also, just to add a note, if you use Aza from Sigma, you can dissolved/reconstitute it in DMSO, which is more stable at around 4 degree (atleast in my hand).

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Question:
New What is the best pipeline presently available for analysis of Illumina HumanMethylation450K beadchip data?
Bit confused with the list of available pipelines right from IMA to wateRmelon.

Bit confused with the list of available pipelines right from IMA to wateRmelon.

By Kunal Dhas · Ganit Labs

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3 How much can we convince the results of methylation study on formalin-fixed, paraffin-embedded tissue (FFPE)?
By Yosep Chong · Yonsei University

Jason Iles · Anglia Ruskin University

Genetic methylation is an enzymatic reaction adding a methyl group to carbon 2 , formalin fixation would occur at the amine group of the cytosine but... [more]

Genetic methylation is an enzymatic reaction adding a methyl group to carbon 2 , formalin fixation would occur at the amine group of the cytosine but this is unlikely as these charges will be interacting with their base pairs and not the solution (in theory)

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4 Do you know a protocol for 5mC Immunofluorescence in mouse tissue?
By Carla Cadena · National Autonomous University of Mexico

Carla Cadena · National Autonomous University of Mexico

I'm using mice, in my tissue 5hmC antibody works perfect. The antibody has been proved in other material and it works, but with the same protocol it d... [more]

I'm using mice, in my tissue 5hmC antibody works perfect. The antibody has been proved in other material and it works, but with the same protocol it does not work un mine

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5 DNA dot blotting and Licor Odyssey infrared detection
By Mikael Niku · University of Helsinki

Mikael Niku · University of Helsinki

Thanks again!

Thanks again!

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Question:
Open Can we cleave E.coli DNA by E.coRI in in vitro conditions?
DNA of E. coli. is modified (methylated) inside the cell, so its restriction endonuclease is not able to cleave its own DNA and treat it as self DNA. ... [more]

DNA of E. coli. is modified (methylated) inside the cell, so its restriction endonuclease is not able to cleave its own DNA and treat it as self DNA. My question is does it (DNA) remain methylated in in vitro condition or not?

By Amit Dadhich · Jayoti Vidyapeeth Women's University

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2 I would like to study differences in DNA methylation on a specific gene promoter of cancer cells. Could you help me with a rapid and simple protocol?
By Hamza Mameri · French National Centre for Scientific Research

Hamza Mameri · French National Centre for Scientific Research

Thanks

Thanks

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Question:
Open How to check stability of valproic acid and 5-Aza-2'-deoxycytidine.
I am working with valproic acid and 5-Aza-2'-deoxycytidine. Perticularly, I wish to look for its impact on functional responses of cells. How I can se... [more]

I am working with valproic acid and 5-Aza-2'-deoxycytidine. Perticularly, I wish to look for its impact on functional responses of cells. How I can see, whether my inhibitors are working or loosing stability?

By Jaydeep Bhat · Universitätsklinikum Schleswig - Holstein

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16 Specific promoter methylation using siRNAs
By Maria Thomas · Institut für klinische Pharmakologie

Abdelhalim Boukaba · University of Valencia

Maria, This an open question and for this reason don’t expect real answers at least from people who may be working on it. I only want to add to Ant... [more]

Maria, This an open question and for this reason don’t expect real answers at least from people who may be working on it. I only want to add to Antoni’s explanations that in mammals, the endogenous RNAi pathway hasn't been dealt with in depth as in other model organisms...It is not clear yet....and negative results are only that until someone demonstrate the contrary. I am not going to say more except that the lack of an Rdp1 homologue in mammals and also in Drosophila may help explain the differences.

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1 Are the TET antibodies (ChIP grade) for HUMAN cells commercially available?
By Gaetano Verde · CRG Centre for Genomic Regulation

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9 Functional significance of intragenic CpG island
By Deepti Kumar · Miami University

Deepti Kumar · Miami University

Thanks all for reply @Abdelhalim thanks for your suggestions. I have also read about intragenic DNA methylation and how it can alter transcription e... [more]

Thanks all for reply @Abdelhalim thanks for your suggestions. I have also read about intragenic DNA methylation and how it can alter transcription elongation efficiency. @ Alan i have looked at the methylation status of the intragenic CpG island in kidney (that does not express this gene) and sperm cells (since it is a sperm specific gene) and i find that it is a differentially methylated region. being hypermethylated in the kidney and hypomethylated in the sperm.

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11 How can an alteration of the promoter sequence of a gene induce an epimutation?
By Sébastien Küry · Centre Hospitalier Universitaire de Nantes

Sébastien Küry · Centre Hospitalier Universitaire de Nantes

Dear Wei, this complex relationship between SNV, miRNA regulation and methylation is also quite interesting. It should indeed concern a more comprehen... [more]

Dear Wei, this complex relationship between SNV, miRNA regulation and methylation is also quite interesting. It should indeed concern a more comprehensive methylation change, as you suggested. Thanks Jon for this additional reference. I will read it rightaway. You’re true Halim. It’s easy to take a shortcut. And when we’re focused on a topic, we have a tendancy to hear and read about it everywhere. It’s also true that it’s often when you really spend time and energy to search something that you find it; otherwise, when you don’t care much about it, you miss it (thanks Lapalice). Anyhow, I’ve seen that several teams focused on the association between genotype and methylation. For further reading, I found a few other relevant references: Quon G et al. Nucleic Acids Res. 2013 Feb 1;41(4):2095-104. Heijmans BT et al. Hum Mol Genet. 2007 Mar 1;16(5):547-54. Drong AW et al. PLoS One. 2013;8(2):e55923.

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4 Does the methylation pattern from whole blood DNA change with different temperatures?
By Silvia Canivell · IDIBAPS August Pi i Sunyer Biomedical Research Institute

John Holloway · University of Southampton

Im not sure though - there are a couple fo recent papers that show you can get whole genome methylation profiles from DNA extracted form blood spotted... [more]

Im not sure though - there are a couple fo recent papers that show you can get whole genome methylation profiles from DNA extracted form blood spotted on guthrie cards stored a room temperature and that these correlate highly with profiles on matched DNA samples from cells stored at -80

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6 HRM Mastermix
By Olivier Switzeny · Johannes Gutenberg-Universität Mainz

Olivier Switzeny · Johannes Gutenberg-Universität Mainz

Thank you all for your valuable input. Since our RTpcr machine has an accuracy of just 0.20 ° C, I´ll try the biorad supermix. The qiagen mix might ... [more]

Thank you all for your valuable input. Since our RTpcr machine has an accuracy of just 0.20 ° C, I´ll try the biorad supermix. The qiagen mix might not produce nice results on our machine, since it is designed for the rotorgene or other machines with an accuracy of at least 0.05 ° C. The protocols are also a bit different, as the biorad mix posses a heteroduplex formation step prior to to the HR melting. I just got a free sample of the biorad supermix and will post my experiences. have a nice week! :)

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4 Is there any commercial kit related to modified streptavidin or avidin?
By Xiaocui Xu · Beijing Genomics Institute

Xiaocui Xu · Beijing Genomics Institute

@ Abdelhalim Boukaba: Thank you for the paper you recommend. It is queit imformative for me. I will try their approach first and contact you if any pr... [more]

@ Abdelhalim Boukaba: Thank you for the paper you recommend. It is queit imformative for me. I will try their approach first and contact you if any progresses.

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3 Protocol for in vitro methylating the CpG island of a gene.
By Deepti Kumar · Miami University

Deepti Kumar · Miami University

Thanks abdelhalim and barbara for your reply I will try this protocol and see

Thanks abdelhalim and barbara for your reply I will try this protocol and see

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20 Amplification of very GC rich regions
By Andrew Beggs · University of Birmingham

Feyzollah Hashemi Gorji · Shahid Beheshti University of Medical Sciences

Yes, it's right, annealing time is dependent to type of region and primer. You can play with it

Yes, it's right, annealing time is dependent to type of region and primer. You can play with it

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Question:
Open Is a CMV promoter suitable for use in myeloid cells?
I've heard that a CMV promoter is not usable in myeloid cells, because of methylation and subsequent silencing. However, I can't find some background ... [more]

I've heard that a CMV promoter is not usable in myeloid cells, because of methylation and subsequent silencing. However, I can't find some background on this. Can anybody tell me if it is ok to use a CMV promoter in a lentiviral vector for myeloid cells?

By Malou Hermkens · Erasmus MC

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Question:
Open Epigenetic modification and host controlled restriction modification:
Is there any evolutionary link between the host controlled restriction modification in bacteria (caused by methylation of bacterial sequences to prote... [more]

Is there any evolutionary link between the host controlled restriction modification in bacteria (caused by methylation of bacterial sequences to protect viral entry) and methylation of nucleic acid sequences in humans to control transcription?

By Guruprasad Gopalakrishnan · PSG Institute of Medical Sciences & Research

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5 H3 and H4 Acetylation and H3K4, H3K9 and H3K27 Methylation
By S. Safi · National University of Science and Technology

Elizabeth Chen · Universidade Federal de São Paulo

Hi, I'm using this technique to evaluate histones modifications (H3K27me3 and (H3K9me3) in gastric cancer

Hi, I'm using this technique to evaluate histones modifications (H3K27me3 and (H3K9me3) in gastric cancer

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9 I want to setup a Bi-sulfide treatment (methylation assay) for my DNA samples? What is the best Kit in this regard?
By Alireza Zare Bidoki · Tehran University of Medical Sciences

David Barker · University of Louisville

I would also follow Karen Reece's suggestion, that you elute with your own water or Tris EDTA buffer with whatever kit that you use. One small issue ... [more]

I would also follow Karen Reece's suggestion, that you elute with your own water or Tris EDTA buffer with whatever kit that you use. One small issue with the Zymo kit was that the elution buffer provided with the kit had something in it that made the absorption spectrum look odd. Company said that it might be oil from the o-ring in the cap on the elution buffer tube. It did not affect performance, but it was an unnecessary cause of confusion.

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12 How to estimate DNA methylation using real time PCR
By Rahul Bhagat · Kidwai Memorial Institute of Oncology

Alexander Dobrovic · Peter MacCallum Cancer Centre

This is a surprisngly difficult question and one with which we are constantly grappling despite considerable expertise in this area. SMART-MSP is a ... [more]

This is a surprisngly difficult question and one with which we are constantly grappling despite considerable expertise in this area. SMART-MSP is a useful alternative to MethyLight if you have access to HRM. MS-HRM works very well but quantitation is relatively difficult for those many loci in which methylation is heterogeneous. We have reviewed the issues related to quantitation of such loci and this is open access like many of our articles.

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Question:
Open Labs providing LC-MS DNA methylation analyses?
I'm looking for an academic or commercial lab providing LC-MS based global DNA methylation analyses as paid service or on collaborative basis. Prefera... [more]

I'm looking for an academic or commercial lab providing LC-MS based global DNA methylation analyses as paid service or on collaborative basis. Preferably in Europe.

By Mikael Niku · University of Helsinki

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2 Which genomic region is a good site for a pos (and neg) control region when looking at DNA methylation in mouse brain (hippocampus, midbrain, cortex)?
By Julia Schulze-Hentrich · Universitätsklinikum Tübingen

Jackie Zhou · Washington University in St. Louis

I have the same question to ask, but not for mouse, it is for rat. I wonder if there is a database/website which has a comprehensive list of all these... [more]

I have the same question to ask, but not for mouse, it is for rat. I wonder if there is a database/website which has a comprehensive list of all these genes? I don't know about fully-methylated or fully-unmethylated genes, but I know there is a database for imprinted genes (hemi-methylated) in different species including mouse, maybe you can take a look at that first: http://www.geneimprint.com/site/genes-by-species.Mus+musculus

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3 Has anybody found a reliable way to employ ELISA-based DNA methylation quantitation kits?
By Francesco Natale · GSI Helmholtzzentrum für Schwerionenforschung

Abdelhalim Boukaba · University of Valencia

Hi Francesco, I think you should perform your experiment as you have planed. I only wanted you to have in mind some parameters. It is always interest... [more]

Hi Francesco, I think you should perform your experiment as you have planed. I only wanted you to have in mind some parameters. It is always interesting if you can find some specific stimulation which is capable to induce global genomic DNA methylation changes. Your next step will be to locate their genomic loci... You can use the Infinium HumanMethylation450 BeadChip Kit as an alternative to NGS. In the past we have used the 27K beads array with negative results. We could find very important data from NGS (still to be published). I think if you can gather some interesting preliminary data you can always find a collaborating lab which has access to the NGS facility and most importantly to the bioinformatics. The last resort is a candidate approach. The literature is full of them

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2 Method of choice to quantify methylation in specific promoters?
By Mikael Niku · University of Helsinki

Mikael Niku · University of Helsinki

Thanks for your comment Alison! Meanwhile, I have been trying exactly the EpiTect kit and it seems to work well. But regarding primer design, I'm stil... [more]

Thanks for your comment Alison! Meanwhile, I have been trying exactly the EpiTect kit and it seems to work well. But regarding primer design, I'm still a bit confused as there seem to be several opinions on this. Wojdacz et al recommend to include one CpG in the primers too in order to compensate for the amplification bias.

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2 Bisulfite conversion with DNA fragmentation. How to avoid it?
By Pavel Prochazka · Academy of Sciences of the Czech Republic

Pavel Prochazka · Academy of Sciences of the Czech Republic

Hi Andrea, many thanks for your answer. The paper looks interesting.

Hi Andrea, many thanks for your answer. The paper looks interesting.

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1 Large difference between two 'biological replicates' (Sequencing data)
By Jackie Zhou · Washington University in St. Louis

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14 DNA (hydroxy-) methylation detection kits: what to look for when ordering?
By Erica Vos · Universiteit van Amsterdam

Erica Vos · Universiteit van Amsterdam

For this kit, page 6, step 2, each DNA sample to be tested is 20 ng (5 uL). This particular kit is checking for DNA methylation (not hydroxymethylati... [more]

For this kit, page 6, step 2, each DNA sample to be tested is 20 ng (5 uL). This particular kit is checking for DNA methylation (not hydroxymethylation).

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About DNA Methylation

Addition of methyl groups to DNA. DNA methyltransferases (DNA methylases) perform this reaction using S-ADENOSYLMETHIONINE as the methyl group donor.

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DNA Methylation

About DNA Methylation

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