Forum discussion on DNA methylation research http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 13:58:56 -0800 360 http://www.protocol-online.org/forums/index.php?showtopic=11685
I am a long time fan of your forum, so I finally decided to get out of the bushes, stop lurking and actually participate in the forum discussions .

I have been reading for a long time the advantages of the fast chip protocol when compared to the regular protocols, so I finally decided to put it in practice. However I am a little confused, do I need to do the column purification (I read in the forum arquives that you use a qiagen column to purify the DNA instead of doing the phenol chlorform extraction)? And if so, which type do you use?

To clarify my doubt:

1) crosslink and harvest cells
2)lyse cells and wash pellet
3)shear chromatine and centrifuge
4)add ab
5)centrifuge to clear aggregates
6)add protein A
7)wash beads
8)add chelex and boil
9)add proteinase K and incubate
10) boil
11) centrifuge and collect supernatant
12) column purification?

Thank you so much to all of you. Your help will be greatly appreciated.
Oh, and thank you jiro_killua I learned to do the ChIP data analysis from you
]]> Thu, 19 Nov 2009 17:32:45 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11685 http://www.protocol-online.org/forums/index.php?showtopic=11569
I'm trying to investigate the methylation status of the promoter. I do the bisulfite modification of genomic DNA using Invitrogen kit, then I succesfully amlify my regions of interest by PCR. I use Taq polymerase in PCR reaction to generate 3' A ends of PCR product. I clone PCR products using TA method into pTZ57R/T. Then transform cells and plate them on ampicilin and IPTG/X-gal medium.
The problem is, that most colonies I get have insert of 100-200 bp less than needed. And it is the most common result. I try checking them by using both colony pcr (M13 primers) and restriction analysis (PvuII) and I get consistently bad results.

Generally I got some plasmids with correct insert and succeeded sequencing them, so that shows my work method is generally right.
But the sucess ratio is unexpectedly low - about 1 out of 100 tested. I checked my plasmid stock. It is not contaminated.

Anyone has any ideas what is going on ? If you can't give any suggestions how to succed, at least write some theoretical considerations about what is happening there.

thanks.
]]> Mon, 16 Nov 2009 02:24:55 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11569 http://www.protocol-online.org/forums/index.php?showtopic=11534 I am completely new in the field of bisulfite sequencing and have some questions concerning my first analysis of the obtained sequence reactions. I have used the DNA methylation KIT from Zymo to convert my gDNA and designed primers for subsequent amplification using MethPrim. Up to that timepoint it has worked fine. Then I started sequencing and received low quality sequences. I have designed internal primer in order to improve the quality. It helped a bit.

Now my questions:
- do I need to do gel extraction on all of them?
- is cloning an option?
- how often do I have to convert the DNA/ amplify in order to confirm my findings.

I have many C-T double peaks which (as far as I understood) could be explained by either a mixture of cells or two alleles differentially methylated. Strange enough: in my case one allele is deleted and I want to check if the other one is silenced by methylation. gDNA has been extracted from cancer cells (assumed to be >90%). So is that an indication that my double peak results from contamination with another sample or is there another explanation?



Thanks a lot for your help.]]> Fri, 13 Nov 2009 13:44:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11534 http://www.protocol-online.org/forums/index.php?showtopic=11408 ]]> Fri, 06 Nov 2009 15:06:52 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11408 http://www.protocol-online.org/forums/index.php?showtopic=11402 Thanks
]]> Fri, 06 Nov 2009 07:42:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11402 http://www.protocol-online.org/forums/index.php?showtopic=11380
The reason I'm asking this is because I want to find out whether we can use FACS to isolate chromosomes and for next generation sequencing without having to sequence the whole genome...

Cheers]]> Thu, 05 Nov 2009 07:42:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11380 http://www.protocol-online.org/forums/index.php?showtopic=11282
I am going to a talk on epigenetics and just had some basic questions about methylation.

What actually causes methylation? For example; if I went running for an hour, genes involved in energy metabolism/mitochondrial biogenesis (AMPK, PGC-1a etc) would become activated/inactivated. Would part of the modification of these genes be due to methylation?

Can all genes be methylated? If not then what does a gene need to have in order to be methylated?

I think that will do for starters. All comments are greatly appreciated.

Superman.

]]> Sun, 01 Nov 2009 06:23:15 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11282 http://www.protocol-online.org/forums/index.php?showtopic=11245
can anyone suggest me a good review paper on the inheritence of DNA methylation? Has anyone looked at the meth profiles between parents and offspring?

Cheers]]> Fri, 30 Oct 2009 00:42:27 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11245 http://www.protocol-online.org/forums/index.php?showtopic=11168
I really need to find a promoter region for a human housekeeping gene that would be suitable to use as an unmethylated control in a MeDIP. I have been looking through commonly used genes such as beta-actin and GAPDH but they seem to have methylated promoter regions. Not sure what is normally done in relation to this, do people just desing primers to avoid the methylated areas? I am finding this hard at the moment so any help would be greatly appreciated.

Thanks]]> Tue, 27 Oct 2009 15:33:47 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11168 http://www.protocol-online.org/forums/index.php?showtopic=11121 I want to detect global methylation using quiagen kit but have problems. After trying in the way the protocol says, I did not get signal in the samples (less than 0,2 O.D) and very low for the methylated DNA control that it is supplied in the kit. I would appreciate very much if somebody has used it and could tell me what can be wrong. In my experiement I used 50ng of the methylated DNA control and the same for my samples. I could try more. Is it also worth trying to use more concentrated antibodies (I diluted them 1:1000, as it is advised in the protocol). For preparing the samples I just diluted DNA in the binding buffer. Is it worth trying to dry DNA before?

Thanks a lot]]> Mon, 26 Oct 2009 11:43:42 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11121 http://www.protocol-online.org/forums/index.php?showtopic=11023 I just entered in the world of ChIP, and I am currently the only one in my lab to dare starting with this technique.
My question is:
After sonication, I wanted to see the DNA fragments range on agarose gel. So I run the RNase-untreated and RNase-treated DNA sample on 1% agarose gel.
I got one thick band in the lane where I did not add the RNase (no visible smear) while a small smear in the lane where I added RNase (thick band disappeared).
Is this thick band correspond to RNA presence?
I am unable to interpret this for the moment.
If any of you could help me, please
thanx
Juni]]> Wed, 21 Oct 2009 09:31:14 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11023 http://www.protocol-online.org/forums/index.php?showtopic=10997
Hi

Please suggest me online software for automated analysis of Chip-Seq data.

Thanks in advance.]]> Tue, 20 Oct 2009 13:05:23 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10997 http://www.protocol-online.org/forums/index.php?showtopic=10978
I have a question: how much should I take PCR product for electrophoresis and what about precentage of the gel?

If DNA after modyfication is so fragile. If I took not enough PCR product I could not see it, could I?








]]> Tue, 20 Oct 2009 05:43:19 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10978 http://www.protocol-online.org/forums/index.php?showtopic=10955
I'm struggling to make sense of my ChIP-Chip visual data. Let me precede all of this by saying I've done numerous controls and tests at the sonication, IP, and array steps, all of which appear to give me accurate data. However, when I visualize the final ChIP-Chip data, it appears that there are too many jagged peaks throughout the genome, including some large, wide positive and negative peaks. Still, there are also some textbook ChIP-Chip peaks there as well.

But here's the kicker - I've done three completely separate biological replicates from culture to array and get the same results, i.e., similar peaks between each array (even those positive and negative peaks that don't look quite right). So if the data is repeatable, is it really noise? Or is it indicative of actual binding? I have read that noise can contaminate an array data set and give false peaks, but also that there is the 'appearance' of noise when evaluating global repressors, and that it's actually correct data.

Specs: I'm evaluating a repressor protein in E coli using Nimblegen K-12 WG microarrays.

I don't have the experience with ChIP-Chip data to make final conclusions about this data, so any help from any of you who may have seen this sort of thing before is greatly appreciated. I've attached a small (~90kbp segment of the genome) pic of the three replicates so you can see the repeatability. Note that this is raw data, before the peak-finding algorithm is applied.

Thanks!

Chris]]> Mon, 19 Oct 2009 07:37:40 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10955 http://www.protocol-online.org/forums/index.php?showtopic=10930
I'm studying DNA methylation changes in stem cells development. I see stem cell development even in the absence of DNA methylation of some important genes related to differentation. Our hypothesis is that, in our context, stem cell development can happen in the absence of DNA methylation but we'd like to carry out an experiment to demonstrate it. We thought interfering DNMTs and check if in the absence of DNMTs stem cell development can occur. Do you know some experiments I could perform??
Thank you so much!!]]> Sun, 18 Oct 2009 04:15:59 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10930 http://www.protocol-online.org/forums/index.php?showtopic=10900
As far as I understand, a nonspecific antibody (Ab) control for ChIP should be raised in the same species as the specific Ab, and should have the same isotype. Taking into account these criteria, I used an unrelated Ab that had nothing to do with immunoprecipitation of modified histones, but matched the criteria for use as a nonspecific control, and my aspecific binding was perfect. Until I discovered that there was a much cheaper solution: ordering a control IgG. But now I have high aspecific binding... This means that, although the unrelated Ab and the control IgG are both suitable for use as a nonspecific Ab based upon the abovementioned criteria, they give different aspecific binding. This probably depends on the method of purification of the Ab...? So my question is: how do you determine the best control IgG for your ChIP experiments? And actually, isn't this aspecific binding control then a rather 'artificial' and not optimal control for nonspecific binding to the constant region of an Ab...?

Thanks for your feedback!

FYI:
- ChIP with anti-H3K27me3 (Upstate): rabbit polyclonal IgG, protein A purified (nice, low Ct values);
- first nonspecific Ab: anti-Lyn, rabbit polyclonal IgG, affinity purified Ab (low nonspecific binding, high Ct values);
- second nonspecific Ab: Rabbit-Control IgG-ChIP grade (Abcam): rabbit polyclonal IgG, purified with affinity chromatography using mouse IgG-agarose (high nonspecific binding, low Ct values)

]]> Fri, 16 Oct 2009 00:38:31 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10900 http://www.protocol-online.org/forums/index.php?showtopic=10833
I'm new in the field of epigenetics and would be very grateful if somebody of this forum could answer my questions

1) After bisulfite treatment, the forward and reverse strand are no longer complementary. Therefore I shoul measure DNA concentration via Nanodrop for ssDNA, right?

2) After BC-PCR I should have dsDNA again, i.e. I can check for a BC-DNA band (or smear via gel electrophoresis using a standard DNA dye containing glycerol, right? Just to verify whether conversion was succesful.

3) I read in some threads that amplicons up to 1kb were amplified! I extract DNA from frozen mouse brain tissue, i.e. brains were taken, frozen and sliced in 200µl slices via a cryocut within 1 week the latest. Specific brain regions were punched using a 1mm diameter needle or whole brain slices were scratched of glass slides. Right after punching or sratching, DNA will be extracted and diluted to 10 ng/µl if possible. I use the Epitect bislufite kit to convert 1µg of DNA into BC-DNA. I try to amplifiy fragments ranging from 150 to 500bp using BC-PCR. Nevertheless almost none of the primers work. I think it is not a problem of primer design. I used BiSearch and spent really a lot of time in designing these primers. Recently I checked my BC-DNA and was quit shocked to see no bands or smear on an agarose gel. I talked to some other PhD students and they gave me the hint to aliquot and store my BC-DNA at -80°C and thaw it just once since the DNA somehow dissapears after freezing and thawing again. Has anybody of you made a similar experience?

4) Right now, I'm going to try my BC-PCR with BC-DNA stored at-80°C using 10 and 20ng, respectively. How much BC-DNA do you use during your PCR?

5) Can you please explain the principle of nested PCR to me?

Thank you very much.

Kind regards.]]> Tue, 13 Oct 2009 02:29:30 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10833 http://www.protocol-online.org/forums/index.php?showtopic=10810
does anyone have any experience with measuring global levels of methylation using QPCR? I know of a paper by Iacopetta, et al. (2007) who uses LINE-1 and SYBRGreen. They use a methylated and unmethylated clone for their standard curve but I was wondering whether we could just use a curve using Sss1 treated and WGA DNA?

Bram]]> Mon, 12 Oct 2009 00:20:33 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10810 http://www.protocol-online.org/forums/index.php?showtopic=10789
has anyone used nimblegen methylation microarrays? any experience to share?

thanks in advance]]> Sat, 10 Oct 2009 13:18:26 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10789 http://www.protocol-online.org/forums/index.php?showtopic=10756
I am trying to design BSPs for a particular plant gene. I am using online tools to design my primers. However, there is a particular stretch in the gene for which the tool is unable to generate any primer set. This region has high CpG % as predicted by CpGplot tool and is a potential target. So, I plan to design the primers manually.

My question is, when I design reverse primers am I supposed get the reverse complement first and then change all the Cs to Ts or is it the other way around?

Any suggestion would be of great help.
Thanks in advance.

]]> Thu, 08 Oct 2009 12:09:22 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10756