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Old 02-21-2011, 12:43 AM   #1
Leeenaaa
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Default ChIP seq - Different DNA size on agarosgel and Bioanalyzer

Dear all,

I'm performing chromatin imnmunoprecipitation and are shearing my crosslinked cells with a Bioruptor. After reverse crosslinking 65C, 5 h followed by DNA purification with E.Z.N.A cycle pure kit (column based) I analyze my Input chromatin (no IP) on a 1 % agarosgel and with Agilent DNA high sensitivity kit. The problem is that these two assays give totally different results. Agarosgel shows DNA library size of avarage 300 bp while bioanalyzer shows an avarage of 2500 bp?!?!

Which assay should I trust? What could be the reason for this difference?

See attached document.
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File Type: pdf agaros vs agilent DNA HS kit.pdf (90.8 KB, 1389 views)
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Old 02-21-2011, 04:40 AM   #2
dariober
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Hi,
Unfortunately I don't have an answer to your question, but I would also like to hear about it. I've seen before similar inconsistencies between gel and Agilent. If you ask me, I believe the gel more than the Agilent. I suspect the Agilent is more sensitive to contamination, overloading, or fragments out of the recommended chip range (and you have a long smear). Did you try to quantify your DNA by Nanodrop? Just to see if the Agilent is getting the concentration right (although the spectrofotometer has also its issues with contamination...).

If you find something... please post!

Good luck
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Old 02-21-2011, 07:18 AM   #3
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Difficult to say what is going on for a number of reasons. But we can probably draw a few conclusions.

First, the amount of DNA in your 2.5 kb bioanalyzer peak is very small -- 0.1 ng/ul. You don't say if the sample on the bioanalyzer is at a different dilution than the sample on the gel.

As you wrote above your agarose gel shows a very intense peak at 300 bp that is not present at all in your bioanalyzer High Sensitivity chip chromatogram--except possibly a tiny blip of a peak at 266 bp.

Here are a couple of possibilites:

(1)The intense "300 bp" peak is single-stranded DNA and/or RNA. But this would require that whatever fluor the DNA High Sensitivity kit uses, does not detect ssDNA and/or RNA.

[Note added 3/2/2012 -- 13 months later: I now know this is not the case. DNA chips and RNA chips detect both single and double stranded polynucleotides, although the intensity is somewhat lower for single stranded.]

If you have enough of your sample, you could run a Nano chip. This will detect both single and double stranded polynucleotides. Alternative you could test the concentration of your sample using double-strand-specific and non-double-strand-specific fluors. (For example, picogreen and ribogreen, respectively link ) If your ribogreen assay detects vastly more nucleic acids than your picogreen assay, this would suggest that your sample is largely single stranded.

(2)That little peak at 266 is really your 35 bp marker. But this would not explain what the big peak at 35 bp is. Still, if you can take a look at the .XAD file containing your chromat using the bioanalyzer software, you could select that peak and set it to be the lower size marker band. Then just see if your peak now agrees with the size on your agarose gel. This possibility seems unlikely to me.

Please let us know what your final result is.

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Last edited by pmiguel; 03-02-2012 at 07:41 AM. Reason: typo
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Old 11-27-2011, 02:29 AM   #4
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Default similar problem here

hey guys,

is there any update about this issue? I'm having more or less the same problem, or at least I think I do.
Untill now I could not get the DNA highsensivity chip to work well for ChIP samples, input or sheared chromatin - but I think that is what the ChIP is made for!?

thanks for any info on this issue!
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Old 11-28-2011, 03:30 AM   #5
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Quote:
Originally Posted by Neuromancer View Post
Untill now I could not get the DNA highsensivity chip to work well for ChIP samples, input or sheared chromatin
What exactly do you mean? Sizing difference between agarose and lab chip? Or something else?

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Old 12-03-2011, 05:32 AM   #6
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Yes, we have exactly the same problem. On agarose gel ist looks like avg. size of 200-400bp. On the Bioanalyzer DNA HS kit it gives a slight smear in that range plus a peak at ca. 2000 as has been seen in the post before (see attachment).
Could it be that the DNA HS kit has problems with sheared DNA?
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Old 12-03-2011, 07:04 AM   #7
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Originally Posted by Neuromancer View Post
Yes, we have exactly the same problem. On agarose gel ist looks like avg. size of 200-400bp. On the Bioanalyzer DNA HS kit it gives a slight smear in that range plus a peak at ca. 2000 as has been seen in the post before (see attachment).
Could it be that the DNA HS kit has problems with sheared DNA?
Maybe it is ssDNA? We ran the RNA ladder standard on a high sensitivity chip (see here:)

http://seqanswers.com/forums/showthread.php?t=12852

with results you may find surprising.

ssDNA would not stain very brightly with EtBr (if that is what you used in your gel...) but it shows up fine on a High Sensitivity Chip. Only problem then is that the ssDNA molecules would need to be ~1.5 thousand nt in length to run as a band of that apparent molecular weight on the High Sensitivity Chip.

So, maybe the temperature of your shearing (sonication?) was not sufficient to prevent some strand denaturation? If single stranded molecules are more recalcitrant to shearing than double stranded molecules that would explain the second peak...

Speculation on my part, obviously...

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Old 12-03-2011, 09:34 AM   #8
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Your agarose gels looks massively overloaded which makes the chromatin appear to run as a small band like you see. Can you rerun some serial dilutions of your input on an agarose gel.

Don't believe me, check out the figure in this protocol and you'll see that when the gel is overloaded the chromatin looks small and well sheared.
http://myers.hudsonalpha.org/documen...%20v042211.pdf
However, their assessment of the gel is 100% wrong.

I've done some work looking at X-linking conditions and shearing conditions with the Covaris so if you give more details about your x-linking, chromatin preparation and shearing conditions I could probably give a better guess.

This might interest you:
http://ethanomics.wordpress.com/2011/11/30/177/

As well as this:
http://ethanomics.files.wordpress.co...ip_covaris.pdf
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Old 12-04-2011, 07:21 AM   #9
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Hi Ethan,
My interpretation of the gel in the HudsonAlpha pdf is that the lower molecular weight DNA is pulling EtBr out of the gel as it migrates -- effectively destaining the gel as it passes. In the case of the highest loadings no EtBr remains to stain the larger (~5 kb) MW "smear".

That assessment may coincide with the description "the gel is overloaded" in the lanes where the high molecular weight smear is not visible.

Then there appears to be another issue where the lower molecular weight smear trends downward in apparent size as the gel is more heavily loaded. That is more the canonical "overloaded gel" effect. Like the gel matrix (agarose?) is failing to retard the progress of the larger numbers of molecules passing through it as effectively as it does a smaller number.

Note that when examining a gel of this sort we are essentially being asked to pre-suppose a number of salient factors:

(1) What "staining" dyes are being used. Eg, EtBr. What concentration. To what components (eg, loading mixture and/or gel, and/or running buffer, or post-run stain only.)
(2) What was the composition of the running buffer, the gel buffer and the loading buffer.
(3) What were the conditions of the run (V, time, length of gel, distance between electrodes.)

Some of these details may be given, but rarely are they all provided. I have my own sort of default presumptions:

Most gels are agarose gels with EtBr in the gel itself, but not in the running buffer or the loading dye mix. The loading dye mix is some glycerol concentration that has been sitting on a bench for over 1 year and has too much Bromphenol Blue in it -- possibly also Xylene Cyanol (also too much). The gel (it will be a minigel most of the time) will have been run just below the voltage that would melt the agarose that composes it. Illumination will be a medium wave length UV transilluminator.

But the chance of all of my presumptions being correct is miniscule.

One plus to use of bioanalyzer chips is that, although secret, many of the parameters are controlled and thus will be the same chip to chip, lab to lab. Also, the protocols offered by Agilent will militate (in some cases successfully) against overloading.

The down-side to using bioanalyzer chips is: it is largely a black box. We may all be using the same (or a similar) black box, but there is much we do not know.

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Old 12-04-2011, 07:49 AM   #10
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Quote:
Originally Posted by ETHANol View Post
Your agarose gels looks massively overloaded which makes the chromatin appear to run as a small band like you see. Can you rerun some serial dilutions of your input on an agarose gel.

Don't believe me, check out the figure in this protocol and you'll see that when the gel is overloaded the chromatin looks small and well sheared.
http://myers.hudsonalpha.org/documen...%20v042211.pdf
However, their assessment of the gel is 100% wrong.
What assessment is that?

By the way, I don't see anything about the protein being purified away prior to running the agarose gel. Does that mean it would still be there, possibly binding the DNA?

I am not familiar with protein binding experiments. Are the proteins that bind the DNA and end up cross-linked to it going to have a positive charge that would tend to drag them back towards the anode? Or do you add SDS to the agarose gels or loading buffer?

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Old 12-04-2011, 11:24 PM   #11
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Quote:
Originally Posted by pmiguel View Post
Maybe it is ssDNA? We ran the RNA ladder standard on a high sensitivity chip (see here

http://seqanswers.com/forums/showthread.php?t=12852

with results you may find surprising.

ssDNA would not stain very brightly with EtBr (if that is what you used in your gel...) but it shows up fine on a High Sensitivity Chip. Only problem then is that the ssDNA molecules would need to be ~1.5 thousand nt in length to run as a band of that apparent molecular weight on the High Sensitivity Chip.

So, maybe the temperature of your shearing (sonication?) was not sufficient to prevent some strand denaturation? If single stranded molecules are more recalcitrant to shearing than double stranded molecules that would explain the second peak...

Speculation on my part, obviously...

--
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Ok, we also ran the RNA ladder on the DNA HS chip and have similar results to yours (5 peaks with completely weird sizes). But that might also be due to different conditions of the gel, dye and migration program on the DNA vs RNA chips...? like you said, it's a black box...
However, I don't see why we should have ssDNA in our soultion.

Maybe a few key features of our protocol will help:
We are using Diagenode's low cell# ChIP kit and X-linking is done with 1% Formaldehyde. Shearing for 25 minutes in the Bioruptor with changes of icy water every 5 minutes. For reverse x-linking we use several ours with prot k @ 55C and then purify DNA with phenol-chloroform.

I don't see where in this protocol you can denature such a huge amount of DNA...

Furthermore, I really do not see that our gel is overloaded (but I will try rerunning lower amounts!). And there are several arguments against the EtBr-pulling theory. First, EtBr runs to opposite direction compared to the DNA, second there should be a lot more EtBr than could actually be taken up by the DNA, third high MW DNA stains a lot better with EtBr due to its lenghth (more space for binding of EtBr).

Still not happy with this Bioanalyzer thing...

So how do other labs check the correct size and concentration of samples before ChIP-seq library prep??
As we don't have a Qbit we need to use the Bioanalyzer to check concentrations after ChIP, I really thought that the DNA HS chips were desinged for exactly that purpose...
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Old 12-05-2011, 11:54 AM   #12
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Quote:
Originally Posted by Neuromancer View Post

I don't see where in this protocol you can denature such a huge amount of DNA...
Okay, I have no reason to doubt you. Except that in combination with the shadowing effect I mention below, it would easily explain what you see. Also, if the DNA was strand-denaturing it might be harder to shear... Still pure speculation on my part.

Quote:
Originally Posted by Neuromancer View Post
Furthermore, I really do not see that our gel is overloaded (but I will try rerunning lower amounts!). And there are several arguments against the EtBr-pulling theory. First, EtBr runs to opposite direction compared to the DNA,
Yes it runs in the opposite direction, and when there is sufficient DNA, it casts a "shadow", binding it up before it reaches the higher molecular weight fragments.

Do you add EtBr to the running buffer as well? Or just to the gel?

Quote:
Originally Posted by Neuromancer View Post
second there should be a lot more EtBr than could actually be taken up by the DNA,
I don't see you have a basis for this claim. How do you know?

Quote:
Originally Posted by Neuromancer View Post
third high MW DNA stains a lot better with EtBr due to its lenghth (more space for binding of EtBr).
But it will see a lower concentration of EtBr because the lower molecular weight smear will have bound much of it.

This is going to be the case unless the gel is stained after running. There are lots of factors in play, but in cases where there is the amount of DNA I see in the smear in your gel I do expect little of the dye to be available to the higher molecular weight (~5kb).

Quote:
Originally Posted by Neuromancer View Post
Still not happy with this Bioanalyzer thing...

So how do other labs check the correct size and concentration of samples before ChIP-seq library prep??
As we don't have a Qbit we need to use the Bioanalyzer to check concentrations after ChIP, I really thought that the DNA HS chips were desinged for exactly that purpose...
I see no reason in this case not to trust your Bioanalyzer result. I think the agarose gel is the one misleading you.

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Old 12-06-2011, 12:11 AM   #13
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Hey Philip,

thanks for the detailed analysis. You might have a point there...

I will try to figure it out and keep you posted about it!
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Old 12-12-2011, 06:06 PM   #14
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Hi!

Not sure if this issue of gel vs bioanalyzer has been resolved yet, but I have some thoughts about this.

1) In your second uploaded pdf, the intensity on the bioanalyzer chip looks very high. If the bioanalyzer chip is overloaded, I have observed the recorded size of the DNA to appear much larger than it actually is (the quantity of DNA causes its migration in the microfluidic chamber of the chip to change such that the chip detects the DNA at a later time than it should, which displays as a larger size). And in addition, not only will this happen if your one sample is overloaded, but if OTHER samples on the chip are overloaded, it will run-over into the subsequent wells, showing up as a high mol weight DNA smear. So, you may want to look back and see if the other samples on the same chip were overloaded.

2) In your second pdf, for the agarose gel image, I actually do see a faint band at the top of your gel. This band could show up as being much "brighter" in the agilent chip because of the way the intensity is measured, maybe? If you look at your quantitation for the bioanalyzer, the software can calculate for you the number of actual molecules you have in a particular size range (use "regions" instead of "peaks"). When you compare the number of molecules in the high mol. region vs low mol. regions, you might find that you actually have way more molecules in the low mol region anyways.

3) When you purify your DNA, do you use any sort of carrier RNA or protein such as glycogen? Is it possible that those carried forward into your bioanalyzer reaction? Those may not have an effect on the agarose gel, but might show up as big sized stuff in the chip.
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Old 12-13-2011, 01:49 AM   #15
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Quote:
Originally Posted by Neuromancer View Post
Furthermore, I really do not see that our gel is overloaded (but I will try rerunning lower amounts!).
Care to bet on that. If I win you'll send me a couple good beers. If you win I'll send you some sun.
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Old 12-16-2011, 06:24 AM   #16
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also another point, not related to the questions here: adding sybr into the gel mix (before it polymerizes) or post staining can have an effect of DNA running differently in agarose.
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Old 01-02-2012, 09:44 PM   #17
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Hi Everyone,

Sorry for being so late on this conversation, but we have seen this quite a bit, and I do have some input:
1. Both gel images shown have been severely overloaded. The gels are also run too fast. You can tell this by the fact the the sides of the lanes and running much further behind than the middle of the lanes. We run 1.5% agarose gels, and run the gel for 3-4 hours at 15-20 volts.
2. Bioanalyzer traces are log based, so a large distribution of higher molecular weigh fragments are compacted into a a much smaller area of the trace as compared to the smaller size fragments.
3. Sybr does effect the mobility of DNA in gels. It is suggested to add Sybr into the loading dye which is then mixed with the DNA samples prior to loading on a gel.
4. If staining with EthBr, then the gel should be post stained.
5. 1.5-2kb peaks noticed on bioanalyzer data when analyzing for chromatin sharing is due to over-cross linking of chromatin. Please take a look at the attached file in which we illustrate the appearance of the 1.5-2kb peak with an increase in cross linking. In our experience, this over-cross linked smear is not reduced further by shearing the chromatin longer.


Thank you

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Old 01-05-2012, 01:00 PM   #18
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Hi Hamid,

In line to your explanation of over-crosslinking of chromatin, I also did some preliminary work on chromatin shearing. I used Advance Analytical (Fragment Analyzer) and Agilent (Bioanalyzer) to analyze the sheared sample. Surprisingly, I see two populations of fragments, one at >1000bp and other at arnd 200bp. Do you think this is becof over-crosslinking or researchers do see this typical behavior with chromatin. Please explain.

Thanks,
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Old 01-11-2012, 08:57 AM   #19
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Hi Smriti,

that is certainly because of over cross linking. we have found that in order to be maximize your chromatin shearing protocol, you must carry out a cross linking time course on the cells you are working with. Cross linking efficiency is certainly cell line dependent. We have customers who cross link certain cells for only 30 seconds because they lose their epitope if cross linked longer.
If you do a smear analysis on your bioanalyzer trace as we did, you will see that over cross linking will effect the percentage of fragments from 150-700bp. If you are doing ChIP-qPCR the percentage of fragments within the library preparation requirement of NGS systems does not make a difference, but if you are doing ChIP-seq it is important to maximize the percentage of fragments within the requirements of the library preparation of your NGS system.

Thank you

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Old 01-18-2012, 08:04 PM   #20
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Hi everyone,

Wow I was happy to find this discussion this morning. I'm also getting huge peaks on the bioanalyzer after doing my chromatin immunoprecipitations, but it's a bit different to what you guys are describing:

I did a basic ChIP to prepare for sequencing (cross-linking with 1% formaldehyde for 10 minutes, sonication with a biorupter for 20 minutes to give fragments of about 200-500bp checked on an agarose gel).

I used magnetic beads with antibodies against histone modifications (plus controls). The input chromatin was 50% of that for the immunoprecipitations. The concentrations on the Qubit are between 0.4 ng/ul to 10 ng/ul. I checked the fragment sizes on a bioanalyzer (HS dsDNA kit) and saw peaks at ~150bp for my input chromatin. But the peaks for all of the immunoprecipitations were up around 3000bp. I'm lost and confused, and would really appreciate some thoughts on this problem

I've attached the graphs: on the left are two inputs, and on the right are two IPs. Also attached my gel after sonication (yes, I know now that it is overloaded and run too fast... ).

Thanks,

Ali
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