DNA Methylation - Science topic

No solution

ran all samples on the q24 in the end at a different lab.

common problem i think…

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.

Hi Tais,

I am having the same problem. Did you solve it?

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.

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

But some of our Indian scientists are good with epigenetics.

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.

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

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

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!

Good luck.

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.

Also check https://www.hindawi.com/journals/ijps/2018/6387826/

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.

You're welcome!

It is better to store your fresh samples in liquid nitrogen. In our lab, we store extracted DNA in a -20 C freezer.

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?

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.

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.

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.

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!

Dear Faaras Iqbal,

Follow the articles below

If my answer was helpful, recommend it.

Best wishes

It could be done by the conventional PCR master mix.

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

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?

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.

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.

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

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

Following

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.

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

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

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

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.

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.

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.

Attached is the Foxp3 HRM TSDR methylation assay standard curve. We do not recommend using WGA after SssI treatment.

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.

NCBI has a tool to convert genomic coordinates from different assemblies. Here is the link: https://www.ncbi.nlm.nih.gov/genome/tools/remap .

Nitesh Kumar Sharma thank you very much...

I may offer to try our GLAD-PCR-assay

We used it recently for the development of epigenetic diagnostic tests.

Thanks, Britta Boserup Thestrup! That is encouraging news!

Do you find the appropriate protocol?

Thank you Liying Yan. The aim was to compare two similar freely available data sets by looking up one CpG in each data set.

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.

When?

12-15 November 2019

Where?

Berlin, Germany

When?

12-15 November 2019

Where?

Berlin, Germany

Thanks for sharing this!

Neha Singh

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.

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

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.

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:

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

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.

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

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

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.

This is a wide subject but I think that those sites will give you a good start.

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!!

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

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.

Do you consider Pyrosequencing Line-1 methylation analysis. There is no need to do standard curve?

EpigenDx has pre-validated Pyrosequencing assays for the rat Grin2a

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.

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.

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.

agree totally with@ Le Minh Thong, you need to do BLAST

regards

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.

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.

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

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.

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.

For the ones who are interested: I decided to go with the article of Zhou and used the 5 base pairs as cut off.

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.