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Old 07-05-2011, 05:58 AM   #1
pmiguel
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Exclamation dsDNA migrates crazy fast on pico RNA chip

FYI!

It appears that dsDNA fragments run on an Agilent Bioanalyzer RNA 6000 pico chip migrate much faster than would be expected given their molecular weight. The result is that dsDNA appears as peaks of much lower molecular weight than the single stranded RNA the chip is designed to assay.



Both panels are the same sample: a double stranded ladder included in the Agilent High Sensitivity DNA chip. Top panel run on the Agilent RNA 6000 pico chip (cat# 5067-1513), bottom panel run on the High Sensitivity DNA chip (cat # 5067-4626).

So the bottom panel gives the correct size of the ladder bands in bp. The top panel gives the apparent size of those same double stranded fragments run on a pico RNA chip in nucleotides.

The ladder was not denatured prior to loading on either chip.

Roughly the same results for another aliquot of the ladder run on the same pico chip at a 1:10 dilution.

I am off to check whether this is documented in the pico chip manual. Links to other places documenting this phenomenon welcome.

For the converse phenomenon (single stranded molecules run very slowly on a double stranded chip) please see my later thread:
http://seqanswers.com/forums/showthread.php?t=12852
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Last edited by pmiguel; 07-20-2011 at 06:58 AM. Reason: added a link to a related thread
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Old 07-05-2011, 06:27 AM   #2
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BTW, I should point out an issue with the above conclusions. Specifically, I hypothesize above that the 50bp fragment is co-migrating with the 25 nt RNA 6000 pico chip marker. While I think this is the case, one might argue that the 7000 bp peak simple does not appear at all on the pico chip and that all the other peaks are therefore mis-identified. I do not think this to be the case, but do not have strong evidence to the contrary at this point.

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Old 07-06-2011, 06:03 AM   #3
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Quote:
Originally Posted by pmiguel View Post
BTW, I should point out an issue with the above conclusions. Specifically, I hypothesize above that the 50bp fragment is co-migrating with the 25 nt RNA 6000 pico chip marker. While I think this is the case, one might argue that the 7000 bp peak simple does not appear at all on the pico chip and that all the other peaks are therefore mis-identified. I do not think this to be the case, but do not have strong evidence to the contrary at this point.

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Cool stuff. I've never run an RNA chip. If you have some other RNA standard and run it on a chip with and without the DNA high sensitive ladder, what is the ratio of fluorescence between the RNA standard and the lower marker in both scenarios? If it's higher with the DNA ladder added then the 50 bp DNA fragment and 25 nt RNA marker are probably co-eluting.
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Old 07-06-2011, 08:12 AM   #4
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Cool stuff. I've never run an RNA chip. If you have some other RNA standard and run it on a chip with and without the DNA high sensitive ladder, what is the ratio of fluorescence between the RNA standard and the lower marker in both scenarios? If it's higher with the DNA ladder added then the 50 bp DNA fragment and 25 nt RNA marker are probably co-eluting.
I do not see an increased height/area of the 25 nt RNA marker peak in that lane. However, there is fairly high variability in the height/area of the 25 nt RNA marker peak. So I do not think that is conclusive.

On the other hand, were the slowest migrating band on the plot above 3000 bp I would have expected the 7000 bp band to show up somewhere.That largest visible band shows up at 35 seconds, whereas the assay ran/collected data for 84 seconds. I zoomed into the first 40 seconds for the plot above. The rest of the chromatogram is basically baseline.

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Old 07-06-2011, 09:30 AM   #5
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Next time you run an RNA chip you can spike in a bit of the DNA high sensitivity marker into a well with the RNA marker as well and see if the 25 nt and 35 bp fragments co-elute. I wish we had RNA chips so I could play around with this stuff too!
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Old 07-06-2011, 09:41 AM   #6
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What happens when you run the RNA gel in the DNA chip and vice versa?
And is there a difference in the high sensitivity versus the regular DNA7500 gel/chip/marker?
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Old 07-06-2011, 09:53 AM   #7
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I believe all the chips are all the same they just put different stickers on them.
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Old 10-10-2011, 02:57 PM   #8
xqxu
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Default RNA-seq

I am planning to make some cDNA libraries for RNA-seq and like to use DIY reagents for cost reason. Would anyone share his/her receipe and tricks with me? Many thanks!
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Old 10-31-2011, 05:44 AM   #9
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Hi pmiguel, I don't exactly understand why you would run dsDNA on a RNA chip? It's not made for running DNA and prone to get the wrong results.
For what reason do you want to do that, what's the property you want to have analyzed?
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Old 10-31-2011, 07:28 AM   #10
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Hi pmiguel, I don't exactly understand why you would run dsDNA on a RNA chip? It's not made for running DNA and prone to get the wrong results.
For what reason do you want to do that, what's the property you want to have analyzed?
I don't want to speak for others, but these chips are expensive and if you had say RNA and DNA samples to run, it would be great if you could run them on one chip and interpret the results rather than run them on two separate chips.
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Old 10-31-2011, 08:29 AM   #11
pmiguel
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Quote:
Originally Posted by Susanne View Post
Hi pmiguel, I don't exactly understand why you would run dsDNA on a RNA chip? It's not made for running DNA and prone to get the wrong results.
For what reason do you want to do that, what's the property you want to have analyzed?
Hi Susanne,
First, it is possible that my ssRNA will contain some double stranded molecules in it. What will these look like? On, for example, agarose gels, single stranded molecules appear to migrate in some (log linear?) proportion to their molecular weight. So that it my "default" expectation. But it is not the case with Agilent chips.

Second, there is actually a use case for running DNA on an RNA chip. For example see:

http://seqanswers.com/forums/showthread.php?t=15064

In brief, there is a potential issue with Illumina libraries where primer/adapter dimers anneal to full length library molecules. These primer/adapter dimers are undetectable using DNA chips (because they remain annealed to the longer amplicons), but are detectable if the samples can be run strand-denatured.

Not ideal, I admit, but works well enough to be of value to us.

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