Science topic

DNA Methylation - Science topic

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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We are measuring methylation at the POMC gene on human DNA.
We are getting good results with out PCR but are consistently having issues with low peak height and baseline drift on the sequencing step.
The option appears to be to increase PCR product though the manual says the maximum is 10ul. We have tried 15ul and this shows some improvement but does not resolve the issue entirely.
Does anyone have experience of the machine and can you put more product in?
Any suggestions much appreciated.
Toby
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No solution
ran all samples on the q24 in the end at a different lab.
common problem i think…
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I have been collecting DNA methylation data for TNBC patients and while I've only been using data produced using Illumina HumanMethylation450 BeadChip the methylation values vary greatly. To be more specific some of the datasets contain values that only range from 0 to 1 while others have values that range from -5 to 7.
I am unsure if the content of the data is completely different or if the data that ranges from 0 to 1 has just been transformed using something similar to a sigmoid function. Any help would be greatly appreciated.
Thank You
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the value range from 0 to 1 is call "beta" value, while the value range from -5 to 7 is probably "M" values. You can easily convert from one to another.
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I am trying to investigate the global methylation with the kit Imprint Methylated DNA Quantification kit (Sigma-Aldrich). However, the results of all my samples were higher than my control. Thus, all samples showed more than 100% of methylation.
Is anyone with the same problem?
We tried different concentrations of DNA but we always found the same: DNA methylation was higher in our samples compared to the methylated control.
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Hi Tais,
I am having the same problem. Did you solve it?
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In many instances, certain DNA methylation activities can be harmful to humans, causing cancers and various disorders. A majority of my research about plant DNA methylation however shows mainly beneficial effects to the plant when the plant is treated with a stressor. Can methylation also be harmful to a plant a lot?
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Methylation is just another form of gene regulation. Harm only comes when the balance of methylation to demethylation becomes perturbed resulting in the silencing of necessary genes or misexpression of genes which have a negative impact. Mammals also benefit from methylation through increased chromosomal stability and the silencing of viral genomes.
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I'm trying to predict age using DNA methylation but I'm not getting the expected amplicon size on the gel image. Please any suggestions?
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A conventional PCR master mix can be used for DNA methylation assay called Methylation Specific PCR. this method uses 2 pair of primer, 1 normal primer, 1for the bisulfite converted one. one of the primers will show more intensity than the other
if the problem is at the amplicon size, what might be wrong is the estimation of amplicon size, blast your primer to make sure the amplicon size is right
if it's right then I suggest you do nested PCR first then proceed to MSP
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Centers in India that offer hands-on training in bisulfite conversion, DNA methylation, expression, and epigenetics are preferred.
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But some of our Indian scientists are good with epigenetics.
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good day! I am a currently researching about epigenetic variations (Mainly on DNA methylation) of a fish that can change habitats (from saltwater to freshwater), my main goal/topic is about osmoregulation and my target tissue is gills. my original plan was to get samples from both habitats regardless of what sex of the fish is. however, most of the literatures that I have been reading collected male only fish. I would be very grateful for your insights.
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The male tiger pufferfish showed higher methylation level (66.009%) than in female (65.027%) and the pseudo male showed an increase in methylation level (65.945%) which is close to methylation level in male after low-temperature induction. This phenomenon was also observed in Chinese tongue sole and tilapia.
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Is it possible that a monoclonal cell line expressing gfp in a stable way can lose or decrease its expression in the course of the passaging (example: after 20 passaging).
Because of for example a methylation of the promoter etc.?
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Hi Thomas,
I think this is possible and in fact I had a similar situation with HEK cells expressing GFP under a CMV promoter. After acoulpe of passages one could see the GFP signal was getting dimmer and it worsen with susesive passages. A similar line with EF1a as driver didn't have this issue. I was told a that time that it was either the promoter was getting metilated and inactivated overtime or that alternatively a regulatory element in the locus where the transgen got inserteg was getting modified and shutting down the expression of neighbouring genes.
Maybe you could check for methylation in your line within the area holding the GFP cassette.
Share if you find out.
Cheers
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Do any of the cells have genomic DNA that is 0% methylated? I found scientific reports indicating that peripheral blood mononuclear cells (PBMCs) may exhibit such properties. In my research, I use bovine material and I would like to make a curve for Methylation-Sensitive High Resolution Melting (MS-HRM) analyses by mixing 0% and 100% methylated DNA. Unfortunately, such DNA for cattle is not commercially available. I think, it will be more difficult to obtain 100% methylated genomic DNA.
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Hi Robert,
I think no cell type contains fully (genome wide) unmethylated DNA. Several cell types may contain reduced levels e.g. sperm cells. If you are interested in methylation of specific markers you may test several normal cell types for methylation to identify useful unmethylated control. If you want to have a generally unmethylated control you can use genome wide amplification kits. However, we observed low levels of bisulfit-treated WGA-DNA after BS-treatment. Potentially caused by the small size of WGA products.
Regards,
Norman
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Hi, I'm doing bisulfite sequencing to study DNA methylation status of my target gene.
I'm using QIAamp DNA Mini kit(QIAGEN) for obtaining gDNA samples and EZ DNA Methylation-Lightning Kit(Zymo Research) for bisulfite conversion.
As protocol from zymo said that 200~500ng gDNA 20ul is optimal condition for bisulfite conversion, I'm using 200~500ng gDNA(sometime 500ng if my gDNA concentration is enough, but sometime I use around 200~300ng if my gDNA concentration is low.)
My problem is I think I'm getting unexpected results.
In my lab I'm the first person to study DNA methylation and use such kind of kits, I'm trying to get same, or similar results from previously published paper before I get results of my target gene.
The gene used in the paper was highly methylated and methylation status of each CpG sites were around 60~90%(mostly 70, 80%)
But when I did sequencing, only 50% were methylated.
Also CpG sites from each colony was all methylated or all unmethylated.
(I mean, I tried sequencing from 8 colonies and results from 4 colonies said all of the CpG was methylated and results from rest of the colonies said all of the CpG was unmethylated-so I'm hard to trust my results.)
I don't think bisulfite reaction didn't work because almost of non-CpG cytosines was converted to uracil.
I think I'm doing something wrong with my expreiments, so I needs some advices.
1. Is gDNA around 200~300ng not sufficient to perform bisulfite conversion? I've heard that too less gDNA amount or too much bisulfite treating could cause conversion of methylated cytosin to uracil. If it's not sufficient, how much would be optimal?
2. Is it common to get only all-or-none methylated result as me?
3. I didn't use RNaseA during extracting gDNA so maybe some RNA will be remaining in my gDNA. Would this be big problem?
4. Are there some other factors that could be a cause of unsuccessful or unreliable bisulfite conversion/sequencing? (There would be a lot, but I want to check if I'm missing something.)
If you have any other ideas or advices to perform successful bisulfite sequencing, I would like to know it whatever it is. Thank you for reading long question.
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Hi!
In our lab, we use the EpiTect Bisulfite Kit (Qiagen) for the bisulfite conversion and we work with very small amounts of gDNA (~20-100ng). Unfortunately, I don't have experience with the Zymo kit, but EpiTect works really well for us. We work with blood samples and cell cultures.
What kind of colonies are you analyzing? Is it the same organism as in the paper to which you compare your data? Gene methylation varies across tissues as well, so you could get different results if you analyze your target region for example in plasma, liver tissue, or saliva.
Maybe you can get some control DNA with known methylation levels, and test your workflow? I see that Zymo has this kind of methylated and non-methylated DNA Set: https://www.zymoresearch.com/products/human-methylated-non-methylated-dna-set-dna-w-primers
Good luck!
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I am looking forward to determining the level of DNA methylation in cell lines and animal tissue samples. The determination of 5mC and 5hmC and DNMT1/3 are considerably important. What are the important differentiating steps for samples processing, for analysis of 5mC, 5hmC through HPLC UV/LCMS.
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Hi,
I need epigenetic data file run through the hovarth clock. DNA Methylation Age Calulator, but did not find any description how it should be done?
Please can you help me to find link to describe the process of running the data?
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The website can be found here: https://dnamage.genetics.ucla.edu
Look at the 'TUTORIALonlineCalculator.pdf' file on the page to understand how to submit your data as a CSV file and how to interpret the results.
Alternatively, the R code is available. I think it's linked to in the supplementary data of Horvath's original paper, if I remember correctly.
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For example, DNA has functional groups such as methyl groups and nanoparticles can interact with them and alter them. I was wondering if we have any technique by which we can quantify them or maybe do a qualitative analysis. Any leads would be great, Thank :)
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detecting DNA methyaltion
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Hi Sakar,
Depends on what your research outcome is and the source of your DNA. For example methylation in the CG content is usually present on both strands (symmetrical) and thus the sense strand sequencing would be sufficient. Methylation in the CHG and CHH context, e.g. present in plant DNA, are strand dependent (asymmetrical) and you may want to consider the anti-sense strand as well for a complete methylation profile of your promoter.
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I have a bed file of DNA methylation, I wanted to know do we analyze sex chromosome data with other sites or not? Any help would be great.
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You're welcome!
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DNA methylation represents a covalent bond between a methyl group and a cytosine. To capture the DNA methylation patterns at a certain point in time, is it sufficient to dry the plant tissue or is it necessary to flash-freeze it in liquid nitrogen?
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It is better to store your fresh samples in liquid nitrogen. In our lab, we store extracted DNA in a -20 C freezer.
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Currently, I am working on an aggressive breast cancer cell line. I intend to restore and enhance the expression of tumor suppressor genes by siRNAs against the DNMT gene and induce apoptosis after treating the cells with siRNAs and subsequently exposed to a chemotherapy drug. I performed the MTT and Annexin V/PI apoptotic assays and I have got weird results.
In MTT assay the conc. of chemo drug with no siRNA was ranged from 100nM to 3200nM and the incubation time was 12 hours. In that experiment, the IC50 was 270nM. But, In cells treated with siRNA and the drug, even the highest conc. of the drug, i.e. 3200nM, did not induce cell death (the ODs were the same for each conc.). To clarify, I reverse transfected the cells with siRNA using RNAimax lipofectamine by Thermo fisher protocol (12 hours incubation time). subsequently, replaced the medium with the serially diluted conc. of the drug in RPMI containing FBS. after 12 hours of incubation, I have proceeded with MTT assay.
In the annexin V/PI experiment, I reverse transfected the cell only with the siRNA. the percentage of the cells in the Q4 quadrant was 90%!! As I understand, the siRNA against DNMT is supposed to induced apoptosis. but the results are showing a completely different outcome.
I have done the same procedure on CMML cells and the results were showing a considerable induction of apoptosis. the only difference was the transfection time, 19 hours against 12 hours.
Any ideas?
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Linna Green and no, I have not tested the transfection efficiency. Because of the sanctions against Iran, I am short in recourses in materials, including lipofectamine RNAimax. I thought if I perform my experiments by the general information in Thermo fisher protocol, maybe it is more efficient. But it seems I was wrong. Do you have any suggestions on how I could optimize the process by using low amounts of lipofectamine?
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Hello, everyone!
It is known that DNA methylation in promoter regions containing CpG islands is responsible for downregulation of genes. How about genes which don´t contain CpG islands in their promoter region? I have been investigating a gene which contains CGs in its promoter region. Based on data from Xenabrowser, there is a negative correlation between methylation and expression levels. However, MethPrimer didn´t identify a CpG island (based on criteria such as GC% content or O/E ratio) in that region. How likely is it that methylation plays a role in the regulation of this gene?
Thank you very much!
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To my knowledge, there are two main ways in which CpG methylation can regulate gene expression.
One of them is by "blocking" the promoter region of the gene in question. The transcription machinery is not able to attach to methylated DNA, thus the gene remains "silenced".
The other option is through chromatin condensation. CpG methylation can be recognized by epigenetic machinery leading to chromatin rearrangements that will eventually turn open (transcriptionally "active") chromatin into condensed (transcriptionally repressed) chromatin.
I don't know exactly how many methylated CpGs are required to block the transcription machinery from making its job neither how many are required to induce chromatin rearrangements through epigenetic machinery. However, I've seen some reports in which methylation of single CpGs could lead to gene silencing. It will depende on the site in which the methylated CpG is found within the DNA sequence. So i´ll think that there could be gene silencing through methylation of some CpGs even when they are not densely packaged within a short sequence of DNA to be considered an island CpG.
To give a more accurate answer to your question, you need to give us more information regarding the analysis that you made with Xenabrowser.
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We know that epigenetic factors are denoted by alterations in DNA methylation, histone modification abd transcription of regulatory non coding RNA such as microRNAd but are there additional epigenetic changes that take place that do NOT involve these mechanisms?
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Nucleosome remodeling and X-inactivation/X-upregulation are a few other examples that might influence gene expression. I have recently addressed the epigenetic mechanisms involved in the pathogenesis of COVID-19. I'd appreciate your comments.
Deleted research item The research item mentioned here has been deleted
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I tried to run this function sitetest to perform Site-level Differential Methylation Analysis using IMA package but I got error message.
sitetestALL = sitetest(dataf,gcase="KO",gcontrol="WT",testmethod ="wilcox" ,Padj="BH", rawpcut = NULL,adjustpcut =NULL,betadiffcut = NULL,paired = FALSE) and I got this error message: Error in wilcox.test.default(x[1:length(lev1)], x[(length(lev1) + 1):(length(lev1) + : not enough (finite) 'x’ observations
Can you help me to solve this problem?
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Hi
I suggest using if and else in lapply.
for example:
if(nrow(coulmn1)> 30) {
x <- with(data, cor(a, b))
}
else {
x <- 0
}
This is a good solution when the number of samples are small.
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I have an inducible expression of the enzyme in a newly derived bacterial strain, and methylation may be affected. I want to test methylation efficiency with a bisulfite kit. The kit is the EZ DNA Methylation kit (Zymo Research, D5001).
Do I need to make a special plasmid with a certain sequence for the bisulfite test, or it works more or less equally for any DNA sequence?
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Dear Joshua Beytebiere , thank you for your kind reply! I was not precise enough in my question, and my wish to keep it short played a bad joke.
I just want to test the methylation state of the DNA in my strain roughly. My plan is:
1. to make a plasmid with a certain sequence insert
2. clone it into a plasmid,
3. transform into bacteria,
4. stimulate expression of my gene, that affects methylation
5. harvest plasmid,
6. make the test with EZ DNA Methylation kit (Zymo Research, D5001)
7. Send 20 clones for Sanger sequencing of the insert.
So I was looking for a special sequence for the insert, that would be my template to test the methylation with bi-sulfite. from your reply I guess the sequence of the "bait" doesn't matter. Thank you!
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I am looking for an online tool to see whether a certain methylated CpG site can affect the expression of a certain gene or not?
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I am trying to predict age from DNA methylation. I am using conventional PCR master mix, but I didn't get my PCR product. Is it due to Master Mix? Should I use HotStar Taq Master Mix ?
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It could be done by the conventional PCR master mix.
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I am currently working on DNA methylation and I needed to determine 1ug of the extracted DNA sample to run my MSAP analysis. I'm stuck on how to get the this done and I need help
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You need to get your extracted DNA (Before bisulfite conversion) and nanodrop it to get a ug/ul concentration. From there you will know how many ul you will need to get Xug/ul
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Hi!
I use pyridine borane to react with DNA. I observed that DNA recovery on the purification stage after pyridine borane reaction is very low, at 10%. Therefore, I would like to kindly ask you about the following:
Does pyridine borane fragment genomic DNA?
Does it affect the efficiency of DNA binding to the column bed during purification?
Does anyone recommend a suitable purification method and tested binding buffers or other suggestions to improve DNA recovery?
Additionally, I have a question, the pyridine borane solution with DNA is shaken for 16h 850rpm, but I wonder if I can mix with vortex before incubation for better distribution of the substance in the solution?
I will be grateful for all suggestions.
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I will only note that Watson and Crick's model is wrong: the two DNA strands are not twisted, but run in parallel. See my article DNA: the Double Helix or the Ribbon Helix?
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Hello,
I am a relatively new in DNA methylation field. I am interested in DNA methylation status of some CpGs sites: two from exons and one from introne. I would like to monitor their methylation status without killing the cells, so conventional sequencing approach do not work. I found the so-called Reporter of Genomic Methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein (Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution https://www.cell.com/cell/references/S0092-8674(15)01098-3 ).
Can someone please explain will the following work or not and why? I plan to make AAV vector with 100-200 bp with the CpG of interest + RGM. These bp also have additional methylated-capable CpGs which correlate with methylation status of the CpG of interest, so if it will report the others it is also fine.
I will appreciate every suggestions or references
Thank you,
Nikita
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Prof. Ayad Ghany Ismaeel , Thank you for the answer. This the paper, which I mentioned in the question. In the article, they provide examples with superenhancers, which are distal regions from coding regions in DNA, as well as with a promoter + the reporter. Both are not coding regions and will not interfere with protein expression. In my case, I am interested in regions which are inside of coding regions. Maybe, if I will add the reporter from the article in protein-coding region, it will interfere with protein expression and will make cells not viable. Thus, I think about a similar approach: to take a region of interest and the reporter downstream and insert it into genome.
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I'm doing an MSc with the Open University researching DNA methylation (5mC and 5hmC) in relation to Alzheimer's disease. I've no connections with a lab, and working from home I am trying to get to grips with the technologies/platforms that are mentioned in the papers I am reading. Could anyone explain the difference between BS and 450K, and if there are benefits in using both in the same study, or is one superior to the other?
Many thanks.
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Bisulfite sequencing could analyze methylation status from a handful of CpG sites from "short" DNA regions or from "all" the CpG sites of whole genome (WGBS). In other words, it is a technique that couples bisulfite treatment of DNA with several DNA sequencing techniques to analyze a few CpG sites or all the CpG sites found in the genome.
Illumina 450K bead array, although its coverage of CpG sites is close to 100% it doesn´t covers all the CpG sites since it analyzes the methylation status of predefined CpG sites. This method is mostly used for big studies where many samples will be processed and a general methylation status of the whole genome is required (e.g. GWAS)
BS coverage: from a few CpG sites to potentially all the CpG sites in a sample.
Illumina 450K bead array coverage: 99% of all the CpG sites in a sample.
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Dear all, I´m experienced in analyzing datasets of gene expression, but for my new project I don´t seem to find a dataset with different time points that is fundamental for my objective. I did find a dataset of an experiment that as the perfect design of what I need but is of data from DNA methylation. It is possible to identify MicroRNAs that putatively are differential expressed based in their methylation state?
Thanks in advanced.
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Thanks Prof. Ayad, I think that this work is going to be pretty helpful. If I have any thoughts I will get back to you.
Best regards
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How can one report the result of an assay on global DNA methylation done using an ELISA based technique.
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Hi Saurabh Mandal, just on your point " If a gene X is METHYLATED in CANCER then mostly it may have an ANTI-CANCER function", this isnt the case. Methylation is provided by the DNMT family on enzymes that provide regulatory and novel methylation in the cell but as of yet do so in a more random fashion (novel) or methylate hemi-methylated DNA (regulation). In cancer, whether spontaneous through mutilations or driven through viral vectors like HPV the methylation acquired is random. When we hit the point where the cell becomes cancerous then yes there is methlyation in anti-cancer genes but also in a host of other genes and seeing as we have cancer then there is little methylation in pro-oncogenic genes.
We cant say because X gene is methylated that it has anti cancer effects.
There has been some evidence of semi-targeted methyltion in HPV 16 infections but more akin to preferential binding of particular sequences when DNMT binds with some of HPV 16's oncogenic proteins
Also in the normal state a methylated gene has reduced function (depending if one or both copies are methylated) but this can be reversed by the TET enzymes and others as part of normal cellular control, not just cancer or disease.
Hope that has been some help
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Hi, I treated the cells with 5-Aza-dc and TSA and analyze a specific gene transcription and found out that the gene was induced due to both treatment(q-PCR) relative to control. Then I preform a western blot and found out that the protein level was reduced. This kind of dis-correlation can happen with DNA methylation and histone actylation inhibitors? can someone give a reference for this kind of dis-correlation?
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I'm trying to analyse the methylation status of the promoter of my gene of interest. I run a few tests to make sure everything is working fine. However, one of my problems is the methylated DNA control shows a methylation percentage of 79-87% range, when it should be 100%.
I think this could be due to some noise, as the non-methylated peak are 5-6 vs 35-50 high of the methylated peaks. Also, the control bases (not present in the sequence) have a tiny peak of 2-5 high, which is clearly noise.
I've been looking around the software but can't seem to find a parameter to set up a noise line so those peaks don't get 'identified' as non-methylated. Is there such a thing? What could I do otherwise?
Note. I know my methylated control is ok as HpaII digestion got a single band compared to a smear of the non-methylated controls. Also, I run methylated DNA that I BS converted and a control that I bought already converted, and both show similar ranges of methylation percentage (always lower than 90%).
Many thanks for your help,
Cristina
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It looks to me like a reagent issue. If your nucleotides are suffering from partial degradation (e.g. if out of date or haven't been properly stored) then when they are injected they will produce a small peak: the phosphate groups released due to that degradation will kick-start the enzymatic chain reaction.
As you noticed, you have a T injection away from the CpG sites that produces the same small peak, so it's not a methylation issue.
Unfortunately, there's no way to ignore or adjust for these peaks because they will influence your methylation measurements. The best option is just to use fresh reagents.
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When we want to study epigenetic effects of bush/wild fires, using an ongoing cohort, we need to focus on a limited list of CpGs. Although necessary, the funding agency does not allow exploratory research. We are considering to include CpGs that are differentially methylation after exposure to smoking (568 CpGs), to PM10 and PM2.5 exposure (~70CpGs), and to stress (?? CpGs).
However, we need a smaller list.
Any preliminary data is welcome.
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Hi Wilfried,
My point is just that most part of the time it's cheaper to do 850K ($250-$350 per sample) than designing a more targeted approach unless you have thousands of samples. So I think it's worth to look at that option if the CpGs you are looking at are covered in 850K. it's easier, faster and cheaper.
but we can also help design the targeted by BSAS or Pyro if you want.
Geoffrey
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Hello,
I work with whole genome bisulfite sequencing and I would like to validate the biological response from DNA methylation regions with gene expression.
My question is about number of samples.
I planned work with 4/6 differents genes and 15/20 brain tissue samples.
My samples are very difficult to get, and I'm asking myself if 15/20 sample are too few for a biologial response validation.
If someone could help with that, I would be very thankful.
Best regards,
Jaque!
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It is a low number and that is a limitation but it is also a known limitation when working with privileged samples such as brain tissue. The key here is to look for data points that show high statistical differences e.g. <0.001 not just borderline (<0.05)
I would also see if the results correspond to what has been shown in previous studies if they exist. Just remember to take the results in the context they are in, doesnt mean it isnt worthwhile but that it is a low number that will either go into future meta analysis with other studies or lead down into larger sample number studies
Good luck
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Hi all!
am gonna to study the methylation status for target promoter gene . I have seen that there are alot of kits can be used such as Zymo kits and Qiagen kits but which one is the best ,also in each one there are different kits . So which one exactly is best for me for studying the DNA from blood samples ?
Also if you kow there is an OPTI-Bisulfite method ? Any one used it ?
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Agree with everyone, I used Zymo's kit myself and never had an issue, really good quality. I used the EZ DNA Methylation-Gold Kit.
Just remember when you are going on to qMSP you will only recover >75% of the imputed DNA so you need to factor that in in some way. I found utilising an internal control gene the best way vs a calibrator that may vary by 25% after conversion. Afterwards I used the formula 2^(ct b actin-ct target)*100
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How polymerase reads ori site where it is already methylated in GATC sequence ?
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According to my knowledge, methylation of DNA is associated with a compact chromatin conformation. This makes DNA inaccessible to DNA polymerases. On the other hand, DNA demethylases remove methyl groups from histones which loosens DNA structure and makes it more accessible to DNA polymerases. Thus, it facilitates DNA replication.
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Hello everbody,
recently, I plan to check the influence of DNA methylation and methylation levels on protein activities in vitro (a protein with both methyl-DNA and unmethyl-DNA binding properties), So I need first try to amplify a specific fragment with different methylation levels.
for an example, 50% methylation levels, each DNA molecule is methylated, and 50% cytosine is replaced by methyl-cytosine occasionally, rather than 50% DNA molecules are methylated, 50% unmethylated (which I can acquire by mix unmethylated and methylated DNA)
As I consumed, If I mix different ratio of dCTP:dmCTP for PCR mixtures (50%), during PCR, 1/2 of cytosine (C) position will be replaced by methyl-cytosine (mC) accasionally. the final products will contain 50% mC.
after, I need to check if the DNA contains different levels mC. the slot blot is used, and anti-5mC antibody is applied to detect the methylation levels.
the unthylated PCR product (no dmCTP) show no signal, while the 100% methylated DNA (no dCTP) show strong signal. but the PCR products amplified with different dCTP:dmCTP ratio show similiar signals (10%, 25%, 50%, 75%) to 100% methylated DNA.
could I conclude that all PCR products, even the one with only 10% dmCTP added, are 100% methylated, all C is replaced by mC.
if not, which methods should be applied to detect the methylation levels.
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The ratio of dC versus dmC incorporated will depend on the preference of the polymerase used to synthesize the DNA. If you wish to assess this ratio reliably, you should hydrolyze the DNA to single nucleotides (e.g. with a mixture of DNase and snake venom exonuclease) and then quantify the ratio dC:dmC by some chromatographic technique (thin layer, paper, HPLC etc.), using appropriate dC and dmC standards.
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Hello everybody, I am new with the meta-analysis in Genome Wide Data so I have this doubt. I have read METAL documentation, which is by far the most used meta-analysis software in both EWAS and GWAS microarray data, but I cannot figure out how would be the input for EWAS analysis. As METAL was originally designed for GWAS, one of the inputs is to provide both the reference and no reference allele. Therefore as EWAS arrays do not rely in allele frequencies but in a quantitative measure, I would like to know how would be the input in METAL regarding this case. Thank you so much in advance for answering this issue (which may be easy, but I certainly do not know)
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Finally I got the answer and is... just do not provide the allele frequencies , the analysis will run fine, reassuring this, here is a Github manual of how to perform an EWAS meta-analysis (https://github.com/ammegandchips/meta_EWAS/blob/master/metal.md) . As you can see the parameters regarding the frequencies and genomic control are off.
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I am doing the age related methylation study of snow leopard. Since there is no commercial kit for me to use as control DNA, I made "fully methylated" by M.sssI with both blood DNA and its WGA product.
For blood DNA, I followed the protocol and incubated for 1hr. For WGA product, I tried 2 types: 1st, just incubating it for 4hr; 2nd, incubating it for 2hr and then purifying the product and replenishing reagents and incubating for another 2 hr.
However, according to the result I got from MS-HRM (see in the attachment), they should different relative signals which implied to different methylation rate (100% blood DNA 1hr > 100% WGA 2hr→purification→2hr > 100% WGA 4hr).
I am wandering whether it is because of the insufficient incubation time of WGA or the poor quality of WGA. I also want to know how to check whether the methyation rate really reached 100% or not. If not, how to improve it.
I am also considering to use synthesized 100% methylated DNA of target region if making 100% methylated DNA from M.sssI is too hard. Since the concentration of target region of synthesized short DNA strands must be higher than normal DNA, I think I need to adjust the input amount of synthesized DNA carefully before apply it. Does anyone has the experience, especially when using MS-HRM?
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Attached is the Foxp3 HRM TSDR methylation assay standard curve. We do not recommend using WGA after SssI treatment.
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Hello, Does any one faced transgene silencing after transfection? I have been trying to get good expression from a recombinant antibody molecules cloned in pUC based vector and transfected in CHO cells. Although the cells were selected and checked for expression the expression is very less and there is no reporter gene available to check the transfection efficiency.
I have recently come across the insulation of the transgene on either side with insulator sequences. so did anyone tried the same? If so can i get any protocol or any paper link to learn how to include insulators for achieving high level of transgene expression?
Thanks in advance.
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Hi Anand, I am not sure about CHO cells. However, we have tested in human iPSCs, where gene expression using lentivirus vectors has silencing issues and heterogeneity in expression. But, when we express genes from AAVS1 safe harbour site the gene expression is uniform and its maintained for several generations.
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Hello,
I have a list of genomic coordinates from differentially methylation regions and a I need to extract from them a list of genes, and I don't want do look one region by one.
Is there a R package or other bioinformatic tool that can help me?
Thank you!
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NCBI has a tool to convert genomic coordinates from different assemblies. Here is the link: https://www.ncbi.nlm.nih.gov/genome/tools/remap .
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Usually when we discuss about epigenetic modifications specifically DNA methylation, we observe hypermethylation of promotor or CpG usually results in gene silensing/ low regulation.
In my case promotor region of this specific gene is hypermethylated but mRNA levels and protein levels are upregulated which results in increased in metastasis in breast cancer.
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Nitesh Kumar Sharma thank you very much...
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Hello,
I am wondering what technology I should use best for the detection of methylated DNA. More specifically, I am investigating the MLH1 gene and was thinking of using either MS-HRM, pyrosequencing, qMSP, MSRE analysis. What would you recommend in term of convenience, efficiency and price?
Many thanks :)
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I may offer to try our GLAD-PCR-assay
We used it recently for the development of epigenetic diagnostic tests.
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Our lab has an abundance of ethanol preserved tadpoles and we were wondering whether or not there would be troubles getting typical bisulfite sequencing data from these samples. I understand the methyl group is covalently bound to the DNA and this might make it robust against degradation in the short term (~10-20 years). Any thoughts or advice would be appreciated!
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Thanks, Britta Boserup Thestrup! That is encouraging news!
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Recent Litterature is little poor concerning DNA extraction and possible possible oxidation occuring during extraction. Some works give some protocols to prevent formation of 8oxodGuanosine while the extraction but oxidation of phenol for example could have an impact also of MetCytosine and change rate of MetC / OhMetC / foMetC and cacMetC.
If someone has tips to extract DNA in order to measure DNA methylation and 8oxodG representing the 'in vivo reality'...thanks in advance !
Vincent
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Do you find the appropriate protocol?
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I have analyzed two DNA methylation data sets:
1) Illumina EPIC
2) Hiseq2500 bisulphite sequencing
I selected one CpG site from the Illumina EPIC analysis and extracted its genomic coordinate on Hg19. For comparison, I want to find the same CpG site in the bisulphite sequencing data set on Hg38. Is this possible?
More details, I tried to look up the exact DNA base, with no luck (made sure to match Hg19 and Hg38 coordinates). I also search in a window +/- some bases around the DNA coordinate, with no luck.
Can it be that the bisulphite sequencing data set simple did not have enough cover? Or is it something else I may have overlooked?
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Thank you Liying Yan. The aim was to compare two similar freely available data sets by looking up one CpG in each data set.
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Hi scientists.... recently I give a treatment to primary fibroblast, and I found that the number of 5mc stained punctum decreased significantly. Anyone have some experience or knowledges to explain this? Thanks!
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The treatment itself might be altering biological mechanisms affecting gene expression and/or activity of 5-mC methylation enzymes like DNMT or TET. You can verify this by measuring gene expression levels with RT-PCR, IF staining for the enzymes, or measuring enzyme activity levels with commercially available kits. EpiGentek’s P-3009 for DNMT activity or P-3086 for TET activity are convenient kits to consider.
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Dear community members,
We have available WGBS data for a species. We used Bismark to map the reads to the reference genome. Unmethylated lambda phage DNA was spiked-in, to estimate the bisulfite conversion error rate. My question is, how can I use the information of this error rate to distinguish truly methylated reads from false positives?
I have seen that many people use a binomial distribution to achieve this, and apply a Benjamin-Hochberg correction citing their 1995 paper (https://www.jstor.org/stable/2346101?seq=1#page_scan_tab_contents).
Is there a tool or an easy way to do this? Excuse my ignorance but I am really new to the field.
Thank you for your time,
Panos
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DNA Methylation Data Analysis Workshop
How to use bisulfite-treated sequencing to study DNA methylation
When?
12-15 November 2019
Where?
Berlin, Germany
Link?
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I have whole genome bisulfite sequencing (WGBS) data for a species without reference genome. Is there any pipeline that I can use to process my data ?
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DNA Methylation Data Analysis Workshop
How to use bisulfite-treated sequencing to study DNA methylation
When?
12-15 November 2019
Where?
Berlin, Germany
Link?
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What I am looking for are studies related to parents' ACES leading to autism in their children via changes in DNA; i.e. DNA methylation.
We have the link from ACES to DNA methylation 
and
We have the link from DNA methylation in the parent to autism in their child https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856164/
However, I have not seen any study going from parent's ACES to children's autism.  It might be there.  I just have not seen it. Thanks.
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Thanks for sharing this!
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I am doing bisulfite treatment on DNA extracted from rat brain and I want to know the treatment has been done completely or no. what controls can I use for that ? Can I use DNA extracted from rat lymphocytes as a fully unmethylated DNA? Can I make fully methylated DNA by treatment a normal DNA extracted with M.SssI enzyme?
thanks in advance
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It would probably be better for you to post this question as a separate new question rather than in this thread. Also you need to provide a more complete description of your experiment. Are you doing Methyl-specific PCR (primers are designed to amplify only bisulfite-treated methylated or unmethylated DNA)?
If that is the case, then whether or not you can rely on your result depends on whether the methylated and unmethylated controls show the right pattern--not on whether or not there is any primer dimer.
If you are using some other approach, then you need to say more clearly what that approach is and provide some images of results if possible.
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I'm having trouble obtaining any pure DNA after the conversion for downstream assays.
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Hi Jennifer, Hi Alrayan
Did you have additional experience with targeted or gw ox-bs from that time to date?
Did the kits work well?
I am going to buy "trumethyl ox-bs module" which is now by tecan/nugen
Did you test it? Do you have specific suggestions?
Many thanks in advance for your preciuos help
Lorenzo
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I know phenotypic differences but now I am interested to know the differences at genetic level e.g. Indels or DNA methylation. Thank you
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Indica and japonica rice are the tow sup species of O. sative. ofcourse there are distinct morphological differencesbetween them. But they are have difernecs at genome level as well as genome size, number of ggenes etc, its better to read the genome sequencing articles for both, it will make you clear Asif Ali . I have compiled this data as assignment for Plant Functional Genomics, but I cant find that file to send you.
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Hello everybody, I am working in a conifer tree species, initially i need to propagate the plant through callus, i have established the callus induction but i struggle to regenerate the plant through callus, so i have planned to regenerate the plant through epigenetic remodeling. can we regenerate the plant through DNA methylation process..? i looked at the protocol for this. i would appreciate if anyone have experience in the plant regeneration via epigenetics. Thank you.
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You may find useful these references:
However, epigenetic remodelling is not something you can manipulate at will, it depends on the cell developmental state, the tissue type, the environmental conditions, and the hormonal stimuli present.
My advice is look for available regeneration protocols for close relatives of the species you want to regenerate and test how these work on your case.
Most trees, including conifers, have extremely large genomes with high duplication and tend to have a large number of transposable elements of various classes. It may be hard to promote plant embryogenesis from callus, and sometimes direct organogenesis is more effective.
An example:
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Hi everyone,
thank you in advance for your help.
Can isohelix genefix be used as a collection method to assess methylation levels in DNA from human saliva?
I.e. would it be ok to keep the saliva at room temperature in the hisohelix genefix buffer for several days? Will this affect the methylation status of the DNA or will DNA methylation be stable under these conditions?
Thank you immensely for your help,
Dr. Antonio Inserra
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Depending on how its fixed in that buffer then yes, if the cells are kept in alive in suspension however there may be some change.
Your best bet though and to have an answer if asked when publishing it to run a control sample, same sample of cells from the same source (cultured or otherwise) and suspend them in the medium. Test one within a few hours and the other in several days or however long you think it will be for the real sample cohort. Then you can compare the results, run in triplicate or similar. You should have a good answer then
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I am trying to quantitate promoter methylation of four genes ( SALL3, FERD3L, BARHL2 and PHOX2A) in breast cancer tissue sample and breast cancer cell lines. Can anyone suggest me which is the best method for quantitation of DNA promoter methylation?
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You can use semi quantitative techniques such as the delta delta ct method or derivation of it or create plasmid controls with specific methlayion levels and use those to calibrate the qMSP and create standard curves to be more accurate depending on your need.
Best of luck, know from experience it can be a pain.
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Previously, I noticed that some protein harbor differential phosphorylated site could distinguish the cancer stage during the cancer development. Similarly, I am wondering the different methylated CG sites at the promoter or CpG island may also present different stages of the cancer. The methylation value of the probes in the same gene is different, which is consistent with the definition of Differential Methylated Regions(DMR). So, here is my question: can we find some specific methylated CGs or probes in the same gene to distinguish the cancer type or the stage? Then we can use the simple probe-based RT-PCR or to find out the cancer and its type or stage.
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Yes, its a newer approach to diagnostics. Cervical cancer is a prime example with HPV driving hypermethylation in the cell. Its being researched by myself and others to be used in a conjunction with screening.
Other cancers have hyper and hypo methylation profiles also, depending on the location and cancer in question. The crux of the matter is no one gene would be suitably sensitive or specific as methylation is both semi targeted maybe and also sporadic in a sense. The best approach is using a panel of potential biomarkers to ensure the sensitivity but the specificity may suffer as a result. It is an interesting field. I have no publications yet but have a few posters on my profile if you were interested and hopefully will be publishing a lot of data in the next few months if you are interested.
All the best
Stephen
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Due to that the DNA methylation and the RNA methylation could both regulate the gene expression, so here is my confusion: does the DNA methylation have the effect on RNA methylation? For example, the gene CDO1 is hypermethylated at the CpG island in LUAD, dose this DNA hypermethylation effect the RNA methylation pattern or level? By the way, these two kinds of methylation has the same effect on gene expression or not? And which is the key regulator to the gene expression?
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1.RNA methylation is dependent upon the methylation motif say ggacc.THere is separate set of proteins called writters ,reader ythdf2 and erasers of methylation of RNA.
2.for DNA methylation it depends upon dnmt1,
3.so say DNA methylated in cpg island and there are proteins called mbd which bind methylation mark ,mbd can also bind up with RNA
And influence the methylation of DNA.
4.so RNA directed dna methylation exist
5.mirna also elfluence methylation of mRNA,
so RNA directed RNA methylation exist
6.dna directed RNA methylation not explored
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I want to see global DNA methylation status of samples. What are the different ways by which i can check this?
After doing bisulphite conversion, what all genes i can sequence to understand level of global DNA methylation?
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MspI/HpaII enzyme digestion in genomic DNA samples following LUMA analysis can estimate the global methylation level (see attached slides). LUMA analysis does not involves bisulfite modification.
If bisulfite modification based method is used, Line-1, Alu, LTR12C, or other repetitive elements can be used as global methylation markers.
If you are interested in getting information in gene level, WGBS, RRBS, and EPIC array are bisulfite based methods. Hope this helps.
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Hi, I'm going to research on mechanisms by which cells differentiate in different directions at the epigenetic level. And I choose WGBS to study DNA methylation. Do you have any suggestions on what does the WGBS data sue for, or any research ideas on my project, or any related articles?Thanks!!!
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I'm studying a gene whose expression is regulated by DNA methylation. Cancer cell lines that express this gene show strong hypomethylation, while cells lines that don't express it show hypermethylation. It's also from a family of genes (LY6 family) that are nearly all regulated by DNA methylation. I've confirmed this methylation profile with bisulfite sequencing.
In cells that don't normally express it (hypermethylated cells), I see a strong (~20 fold) increase in expression following radiation treatment. I hypothesized that this was due to changes in methylation. But when I did bisulfite sequencing on irradiated cells, I see that they are still methylated.
Is there any mechanism (besides methylation) that can explain this? I'd really appreciate any help! Thanks!
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Thanks Andriy and Rejane. I'll try again with new samples (the DNA I used was quite old) and see what I get then. If that doesn't work, then I'll consider looking into Pol 2 and other TFs and promoters. Thanks!!
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I am working with DNA methylation currently. I am creating hypomethylation condition in the cells. so how to confirm that the hypomethylation has occurred.
sequencing of only LINE-1 gene will be sufficient to see the global methylation?
or what are the different methods to study global DNA methylation?
Kindly suggest.
Thank you
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Matthias R. Schaefer Dear Sir, I am not Planning my experiment here. According to me, RG is the general platform to discuss science. Hence, I am asking my genuine doubt. if you have any concern regarding my question, please message me before stating irrelevant comments.
Thank you
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I am trying to monitor the methylation statuses of a certain set of genetic loci in glioblastoma cells following a treatment protocol. Can anyone please suggest some easy-to-use assay kits to analyze the methylation levels at certain genetic loci?
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Hi
As you asked for a quantitative result I would suggest to consider either a methylight assay or to go for Pyroseq. The latter one has definitely advantages.
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So these few days I was working with ACTIVE MOTIF Global DNA Methylation LINE-1 Kit, but I have no results because standard curve has a weak signal. For example, the level of "Standard 0" should be at least 0.300 - 0.450 but my result was 0.249. Level of "Standard 100" should be 1,300 - 1,900 but my result was 0,552. I tried it a few times and results I get is almost the same. I also tried to troubleshoot it with Instruction Manual but it didn't help me.
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Do you consider Pyrosequencing Line-1 methylation analysis. There is no need to do standard curve?
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Hello everyone
I would like to know if someone has a validate primer for rat Grin2a to do DNA methylation by pyrosequencing.
Thank you so much for attention.
Yours faithfully,
Camila M Loureiro
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EpigenDx has pre-validated Pyrosequencing assays for the rat Grin2a
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I am searching the literature for an article about DNA methylation signatures in breast cancer. I am finding a lot of articles showing comprehensive analysis between different subtypes of breast cancer, but I did not find one showing ALL the cpg sites that were differentially methylated in tumor versus normal-like breast tissues. If anybody can help, I will be grateful.
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Dear Zainab Awada,
I'm not sure that you can find one article that includes all methylation sites associated with breast tumor development/progression. Try to look through the articles listed below. Hope they would be helpful.
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Dear scientists,
At the moment I am analysing the RNA-seq, ATAC-seq and Methylation datasets of the same mouse-derived cell-type. Methylation was done not as bisulfite sequencing, but as an enzymatic enrichment for DNA fragments (after sonication), containing methylated CpGs with subsequent DNA library prep. It results in ATAC-like peaky view on IGV, so I focus not on the particular cytosines, but on the overall hyper/hypomethylations of promoters, etc.
I have found, that in some cases the Methylation peaks follow the exon structure, and quite often the regions, which were methylated, were not accompanied with Tn5 accessibility on ATAC-seq.
Do you have an idea why introns are not methylated, in contrast to exons? Is it correct to say that the regions of chromatin, which are closed, tend (not?) to be methylated? Does DNA methylation decrease the Tn5 cutting efficensy?
Thank you!
Best,
Michail
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For the differential methylation profile between exons and introns, I would be very careful for the interpretation and double-check that is does not come from some kind of contamination (RNA...), but apparently it's a known phenomenon and it relates to splicing mechanisms, see:
As for other intergenic regions, it's quite well known that DNA methylation recruits the PRC2 Polycomb complex that adds the repressive H3K27me3 histone mark and induces a compaction of the chromatin. It's not so surprising, therefore, that your methylated regions are not detected by ATAC-seq, as they are probably in a less open conformation and less accessible to Tn5 (I refer to your first statement in the 2nd paragraph of your question, but in your second question you seem to have made a confusion between closed or open). I would say that methylated regions tend to be closed, and open regions tend to be unmethylated.
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I am interested in communicating with anyone who is performing single cell / single gene DNA methylation assays. Thanks.
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Hello Dr. Karpf,
I hope you are doing well. One person I know of (previously a colleague, now a PI) works on single cell cancer system biology. The website is http://www.birtwistlelab.com/ if you are still looking for someone.
Best.
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I need to known a DNA sequence for a gene in the plant(V.faba) in order to design primer for PCR for DNA methylation purpose analysis.However I only had the mRNA seq and AA seq for the gene in this plant. What should I do
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agree totally with@ Le Minh Thong, you need to do BLAST
regards
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So, I have read quite a lot of papers that have studied the structure of DNMT3A and its mechanism; however, I couldn't really understand the mechanism fully. Hopefully, you guys can help me understand :)
Based on what I have read, DNMT3A works as a heterotetramer (DNMT3L-DNMT3A-DNMT3A-DNMT3L). Also, based on two papers published in Nature by J. Song and X. Cheng, the two DNMT3A active sites target CpG sites that are separated by approximately 10 to 14 base pairs apart.
So for example, if the DNA sequence is
_________1__________________2_________
5'-CATGCGTTCTAATTAGAACGCATG-3' (sense strand)
3'-GTACGCAAGATTAATCTTGCGTAC-5' (antisense strand)
One of DNMT3A active site will methylate CG (1) and the other DNMT3A active site will methylate CG (2).
Now my question is whether this DNMT3A methylates only the sense strand CG or only the antisense strand CG or both.
Also, if only one of my DNMT3A has a mutation which results in inactive form of DNMT3A (meaning formation of DNMT3L-inactive DNMT3A - active DNMT3A- DNMT3L), what will happen?
Should I expect either CG (1) or CG (2) methylated but not both?
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Dear Jihoon Han;
For the first question, I think both strands would be methylated.
For the second question, only the mutated version would not be inactivated that is bound to other inactivated points i.e. not both sites.
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DNA structure and activity can change with environmental factors. But does experience such as sexual abuse create significant biological effects on DNA?
These type of researches are so important in detecting crime.
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Just like environmental factors, psychological factors may have an impact on gene expression and its regulatory mechanisms but the extent of impact would be depndent on multifactorial aspects and would need to be ascertained in a case to case basis.
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Hi!
We are planning to assess DNA methylation profile of 100-200 cells using bisulfite sequencing on the NextSeq500 instrument. However, the kits designed for single cells (Accel-NGS® Methyl-Seq DNA Library, Pico Methyl-Seq™ Library Prep Kit, QIAseq™ Ultralow Input Library Kit) produce genome-sized libraries that are better to sequence using more high-throughput platforms such as HiSeq or NovoSeq. Other solutions, e.g. TruSeq® Methyl Capture EPIC Library Prep Kit, generate enriched (not so big) libraries that can be sequenced using NextSeq but require a lot of input DNA.
Does anybody know solutions for methyl-seq library preparation and targeted enrichment using a low number of cells? Other words, we need a kit that is similar to TruSeq® Methyl Capture EPIC Library Prep Kit but is designed for a few cells.
Thank you in advance for your suggestions and opinions!
Best wishes,
Evgeny
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Hi Evgeny
I stumbled upon your question looking for the answer to my own. Want to know if you found the solution how to assess DNA methylation profile from as few as 500 cells max. Any suggestions would be highly useful.
Regards
Chandra Shekhar
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Hello all,
I am looking into using DNA methylation to test whether patients have accelerated aging.
I have seen researchers use Hannum's weights for CpGs in order to calculate accelerated aging in some patient populations.
How do we account for the standard error? Where can I find a pipeline for this calculation?
Thanks!
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You can also do what Bryant M Schultz said, but on top of what he said, take into account as well the tissue you are studying. Hannum's signature is built from blood samples. Horvarth's signature, on the other hand, was build from a pool of multi-tissue samples.
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Dr. Magnus Nordborg recently pointed out in a presentation that is no evidence that methylation is involved in either development or adaptation and there is still little evidence of the involvement with gene regulation.
Also, the paper "Epigenomic Diversity in a Global Collection of Arabidopsis thaliana Accessions" (Kawakatsu et al. 2016) showed methylation in Arabidopsis strongly correlated with geography and climate of origin.
It seems that environment controls both methylation and gene expression but there is not necessarily a cause-effect relationship between these two.
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I support the comments of MR Schaefer and I can contribute that epigenetic changes also interfere in the behavior of transgenic plants in field conditions.
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I have DNA methylation data from the Illumina EPIC beadchip (information on 850 000 CpG site). Since SNPs can influence the methylation results, it is advised to filter CpG sites located close to SNPs. The question is how close.
I read that 10 base pairs is commonly used. But in the article of Zhou (2017, Comprehensive characterization, annotation and innovative use of Infinium DNA methylation BeadChip probes) it is noted that the 10 base pair distance is too stringent, since >50% of the EPIC probes carry SNP annotations with a distance < 10 bp. They advice to use a distance of 5 base pairs.
Do you agree with this?
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For the ones who are interested: I decided to go with the article of Zhou and used the 5 base pairs as cut off.
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Hello!
I'm using entropy to measure dispersion in DNA methylation values.
However, I'm not sure which statistical test I may use to determine if the differences that I observe in entropy values between two groups are significant.
Thank you for your answers.
Robin
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Wikipedia may be of use
Although this gives the variance it is not clear from what you have said that the variate is normally distributed
A bootstrap may be a better way of assessing the distribution of this statistic on your data set.