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Old 07-31-2012, 04:15 PM   #1
eab
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Default odd total RNA pattern bioanalyzer

has anyone seen a pattern like this in their total RNA, or know what it might mean?
this RNA was isolated from human T cells using Qiagen RNEasy, dissolved in H2O120731 EB RNA.jpg
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Old 08-01-2012, 04:32 AM   #2
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In all likelihood it means your RNA is degraded. However last month we saw a sample that was isolated using Qiagen RNeasy-mini that had what I took to be an odd pattern of degredation:


or, with the sizing turned on:



Not identical to your plot, but does contain what appears to be a non-standard rRNA degradation. The investigator said they were using this kit. Is that the one you are using?

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Old 08-01-2012, 05:33 AM   #3
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Your trace looks a bit like the 'Noisy peak' described in the troubleshooting guide (http://www.genomics.liv.ac.uk/animal...F/AGILENTM.PDF). I'd try giving everything a good clean and trying again....be sure there's nothing on your bench that might be causing vibrations.

JPC

Last edited by JPC; 08-01-2012 at 06:50 AM. Reason: my terrible grammar
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Old 08-01-2012, 06:01 AM   #4
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Hi JPC,
Is your recommendation directed to me or eab?

In my case, the images I included were the 2nd time the samples were run. Here is the first time:



I have a 3rd run as well, if requested... We nearly always include an RNA sample of known good quality on each nano chip and it looked fine in each case. Our bioanalyzer is on a sturdy bench and not touching anything but the floor. We are in a sub-basement so vibrations are not generally an issue.

I can't speak to the details of EAB's run, but the shape there is not really similar to the "noisy electropherogram" example nor "broad peak". The bell shape is striking. But repeating the run -- especially after giving the sample a hard spin (in a microfuge, >10,000g for 5') and pippetting near the surface immediately afterwards to avoid particulates -- might be worth it.

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Old 08-01-2012, 06:19 AM   #5
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Sorry for not being clear, my comments were directed to ead. Obviously this is subjective but I do think there is a similarity to "Noisy Peak", though ead's trace is not as pronounced as the one shown in the manual.

Either way I would suggest a re-run. I have seen odd traces 'disappear' before now and if it is degradation you may see this develop hence confirming the 'diagnosis'.

Last edited by JPC; 08-01-2012 at 07:25 AM.
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Old 08-01-2012, 06:38 AM   #6
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Really? Here is the picture from the Agilent manual you cite ("Agilent 2100 Bioanalyzer Troubleshooting and Maintenance Guide for Molecular Assays", p72) :




I don't see the resemblance.

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Old 08-01-2012, 07:00 AM   #7
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Yes that's the one, I feel there is some similarity (not a huge amount) and you don't....I don't understand what the problem is?

ead is more than welcome to follow your advice and I have no reason to suggest that it isn't correct.

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Old 08-01-2012, 06:24 PM   #8
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Default genomic DNA

I've decided that this is genomic DNA contamination. I think I see two ribosomal peaks flanking that big bump. Sad to say, I know what degradation looks like, and that bump is too high MW to be degraded RNA. Perfect for normally-distributed fragments of human chromosomes sheared in spin-column purification.

Check this for comparison. http://www.chem-agilent.com/pdf/5989-0991EN.pdf

So now what should I do if I want to make mRNA libraries by standard Illumina protocol? Just go ahead with oligo-dT dynabeads? DNAse-treat? Something else?

Thanks!
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Old 08-02-2012, 02:34 AM   #9
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So now what should I do if I want to make mRNA libraries by standard Illumina protocol? Just go ahead with oligo-dT dynabeads? DNAse-treat? Something else?

Thanks![/QUOTE]

My advice would be to DNase treat, it it is DNA contamination you run the risk of carrying some of it through library prep.
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Old 08-02-2012, 04:12 AM   #10
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So you think I should DNAse treat even though I'm going to do poly-A purification?

If I do DNAse treat, will 94 degrees for 6 minutes (which I do for RNA frag anyway) be sufficient to inactivate the DNAse, or do I need to do additional inactivation? I'm worried about carrying the DNAse through and chewing up my library, but I don't want to do too much heating of the RNA before it's purified, either.
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Old 08-02-2012, 05:55 AM   #11
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No, you want to DNAse treat, then column purify. Zymo sells kits with DNAse and the columns for cleaning up the reactions package in one kit.

By the way, I have a hard time believing that is genomic DNA. Genomic DNA will tend to be >50 kb unless treated very roughly. So I don't see any reason for it to run as a curve of that shape around that size. Could be wrong, though. Those RNA chips have some odd properties.

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Old 08-02-2012, 06:34 AM   #12
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OK, thanks Phillip, I appreciate your sharing your thoughts.

Why won't oligo dT purification on dynabeads be sufficient? Some DNA may copurify, but I would guess that most shouldn't. Any that does get through appears to be pretty large (running between the ribosomal RNA peaks). Will be tougher to incorporate it into a library and quite tough to cluster, no?

For that matter, won't oligo-dT dynabeads take out DNAse, too, if I choose to use it? I do two rounds of binding to the beads with an elution in between.

thanks
eb
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Old 08-02-2012, 08:01 AM   #13
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We had a set of samples that were not properly DNased and the DNA carried through the library prep and was identified when we mapped the reads and found both genome and transcription coverage. My advice is always spend the time on getting the best library prep possible as it can be a very costly mistake not to.

To echo pmiguel I would highly recommend the Zymo columns we have done a head to head test of RNA columns with some of our species of interest and the Zymo columns consistently come out on top. In fact where there is a precipitation step we substitute a Zymo Clean and Concentrate column and have been getting much better final yields.
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Old 08-02-2012, 08:16 AM   #14
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two votes in favor of DNAse -> i will DNAse.

now what about relying on two rounds of dynabeads oligo-dT purification, plus 6 minutes at 95 degrees to inactivate residual DNAse

i really don't want to do more columns because there are many samples and the amount of RNA is very low to start with

thanks!!
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Old 08-02-2012, 08:23 AM   #15
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just to clarify, this is what Ambion says about its DNAse I

"Heat Inactivation of DNase I Some protocols suggest heating at 75C for 5 min to inactivate DNase I [5]. A 10-minute incubation at 75C completely inactivates Ambion DNase I at a concentration of 0.1 U/uL. If this is the preferred method of inactivation, add EDTA to a final concentration of 5 mM before heating"

seems to me that 6 minutes at 95, which is done for fragmentation of RNA anyway, will eliminate any DNAse activity not removed by Dynabead purification.

anyone disagree?
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Old 08-02-2012, 11:15 AM   #16
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Okay, I think you are getting ahead of yourself here. The simplest explanation for the result you posted initially is that your RNA is degraded. But it looks strange enough that it would be worth rerunning it. But for TruSeq DNAse treatment is recommended, so you might as well do that first -- if you clean everything up with a column. Then you can take a look at it on a chip again.

But, the most likely outcome is: degraded RNA, start again.

One step off the path as designed by Illumina, I'm willing to chip in suggestions. Two steps -- okay I'm still listening, not sure anything I say is going to be of much help. But you are 3 steps out now and on your own.

Let us know if it works, but you (1) have circumvented the normal QC step by declaring its failure was the result of genomic DNA in your sample -- which would not be there is you had not (2)also skipped a step asked for by the protocol by not DNAsing and now you (3)think that the fragmentation step should be sufficient to inactivate any DNAse still around after the polyA+ isolation. So, I can't say it will fail, but I don't like your chances.

The big problem is that the most likely failure mode, high 3' bias caused by low intactness of your total RNA, will not be obvious until you complete mapping back to a reference genome.
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Old 08-02-2012, 11:48 AM   #17
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One more thing to factor in: these are irreplaceable human samples. There is no starting again.

So...I need to squeeze the best possible data out of them. If these data don't live up to my wildest dreams, I can accept that. 3' bias? Not the end of the world. I gain nothing by throwing the samples away.

Another consideration I have not made totally clear: there is very little RNA here. I do not like the idea of column purifying for that reason.

Definitely a mistake not to do on-column DNAse digestion, no argument from me there. I use RNAzol for my extractions, and this kind of contamination has not been a problem for me before. In this case, I have been trying to help a colleague make libraries from cells sorted into RLT buffer. Should it come up again, I shall know that the DNAse treatment is mandatory.

But, back to these samples. To me, there is just no way that a hump of material between two intact-looking rRNA peaks can be degraded RNA. If degraded material is going to pile up in a single hump, that better be a low-MW hump, not several Kb in length. I am sufficiently convinced by 1) the absence of a solid alternative explanation and 2) the similarity to the pics I sent out previously (see http://www.chem-agilent.com/pdf/5989-0991EN.pdf, especially page 3, item B) that I am going to proceed as if it's genomic DNA. And yes, I do think that the 95-degree, 6-min fragmentation step should be sufficient to kill any DNAse that manages to persist through two rounds of dynabeads purification (which to my mind isn't so different from a column purification, anyway).

But of course I could be wrong. The plan is to pool aliquots of residual material I had frozen separately for the Bio-A, and make one single library according to my made-up method. I will post results either way.

Thanks all for your advice,
Eli
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Old 08-17-2012, 10:21 AM   #18
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Default Dynabeads and DNAse?

I don't think it is a good idea to have active DNAse during the dynabeads procedure- aren't the oligos made of DNA? If so this will ensure that you don't recover any RNA, as the DNAse will degrade the oligos so there will be nothing for your RNA to hybridize to. Also, you run the risk of seeing lots of oligo dT in your final library. Maybe I missed something?
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Old 08-18-2012, 05:54 AM   #19
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nope, don't think you missed anything. yeah, the "d" in "oligo-dT Dynabeads" is, like, the same d in DNA. embarrassing.

thank you!
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