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Old 04-10-2017, 12:15 PM   #1
renuka22
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Default RNA degradation?

Hello,

PFA the electropherogram image of RNA degradation on bioanalyzer. There were no peaks from samples which I can conclude as no RNA or RNA has degraded completely. But the RIN value is 3.4. So shouldn't the gel still show some degradation peaks. All my samples show this kind of graph. I am confused as to why is this happening and all the concentrations are between 200-300 ng/ul. I am wondering where did all the RNA go? Anyone having an experience in this I request you to please share your opinions.

Thanks
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Old 04-11-2017, 04:42 AM   #2
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I am confused as to why is this happening and all the concentrations are between 200-300 ng/ul.
Hi Renuka22,
You are giving us almost no information to work with -- for instance what assay told you that you have 200-300 ng/ul? What is your source of RNA (organism and tissue)? What type of preps did you do? How many and what were the RIN scores of the other preps? What instrument is that chromatogram from? (Probably a TapeStation, but who knows?)

If I had to make a blind guess (and that is about all I can do with what you give us here), my guess would be that you used UV spec to measure concentration but your samples were phenol contaminated.

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Old 04-11-2017, 09:13 AM   #3
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Hi,

Sorry about the missing information. I checked on nanodrop and it showed concentration in the range of 200-300 ng/ul. I am working on rat primary retinal ganglion cells and isolating polysomal RNA. I used the TRIZOL method and since TRIZOL inhibits RNase, I don't understand how can the samples degrade. I had total 6 samples. So when we sent the samples for sequencing, 2 samples gave RIN of 1.2 (Please find attached the bioanalyzer data from the company) while others showed N/A, so the company sent us the samples back. Then I again tested it in another lab using tapestation 4200 and 4 samples showed RIN 3.2-3.4 and two samples showed N/A. So I don't know what should be the conclusion of this?
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Old 04-11-2017, 12:14 PM   #4
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Trizol is an excellent source of phenol contamination.
I'm sure you understand that measuring the absorbance of a sample at 260nm in no way proves that that absorbance derives from nucleic acids, right?
Did you check for the hallmarks of phenol contamination of your sample? That would be a higher absorbance at 270nm than at 260nm.
See my post here, for more details about various sources of UV spec absorbance in biological samples.

With chromatograms like the ones you have posting it is difficult to infer anything about the quality (or even presence) of RNA in your samples. But if you are isolating polysomal RNA from "rat primary retinal ganglion cells", I can't imagine your yields could be anywhere close to 200-300ng/ul of RNA unless you are pooling cells from dozens of rats.

This would support the possibility that your sample is phenol (trizol) contaminated and that your yields are much, much lower than what you presume.

If you could post your nanodrop trace, it would be trivial to diagnose this issue.

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Old 04-11-2017, 01:05 PM   #5
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Hi,

I think you are absolutely right. There is a peak at 270nm seen on nanodrop. Is there any way I can clean up the samples with a kit. Will that help? What can be a good solution to this problem? Will I need to do the isolations again?

Thanks
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Old 04-11-2017, 01:10 PM   #6
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Here is the nanodrop image..
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Old 04-12-2017, 02:55 AM   #7
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Okay, at this juncture, your real problem is that you don't know what concentration your RNA is actually at. Secondarily you have some Trizol (both phenol and some guanidium salt at 230nm) remaining in your prep.

Yes you can clean up your samples. I'd recommend an RNA clean-up column from Qiagen or Zymo.

But you might want to check the RNA concentrations on a fluorimeter (eg, Qubit) first. Fluorimetry is much less confounded by various common contaminants of RNA preps and should give you an answer.

Although the bioanalyzer results look really wacky -- so maybe doing the clean-up first is the way to go.

Again, given the source of your RNA, I would not expect your yield to be very high. So a nanodrop may not be sensitive enough to detect it. You might need to go to fluorimetry.

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Old 04-12-2017, 08:05 AM   #8
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Ok got you. I think it would be better to do a clean up first and then measure concentration. Can the DNAse also be a source of degradation because I have not done the DNase treatment? I don't have an experience on RNA work so these things are difficult for me to interpret..

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Old 04-12-2017, 08:28 AM   #9
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DNAse itself should not degrade RNA as long as it it pure. But if your samples have residual RNAses in them from the cells from which they derive, the during the DNAse incubation, those RNAses may well be active. RNAse inhibitor works against some RNAses.

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Old 04-12-2017, 09:06 AM   #10
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Ok. Thanks. Do you have any idea why are the Zymo columns so cheaper than Qiagen. Which one is good? Zymo also has some clean up kits with DNAse in those. Do those work good?
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Old 04-12-2017, 10:12 AM   #11
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Ok. Thanks. Do you have any idea why are the Zymo columns so cheaper than Qiagen. Which one is good? Zymo also has some clean up kits with DNAse in those. Do those work good?
No idea why the Zymo columns are cheaper. They work well. We use them sometimes.

The DNAse/RNA clean-up ones work too. Although people complain of yield issues. Some of the qiagen columns allow the DNAse treatment to be done on the column. That has the advantage of staging the column purification (which would tend to remove proteins like RNAses) before the DNAse treatment.

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Old 04-12-2017, 11:27 AM   #12
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Is it a good idea to do DNAse treatment because I got to know that the yield I will be getting from this tissue after clean up will be around 20ng/ul. I am planning to do a polyA isolation later and RNA seq. In the long run,is the DNAse treatment be absolutely necessary? In short, is it required for samples which will be giving very less yield?
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Old 07-15-2017, 09:20 AM   #13
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Originally Posted by renuka22 View Post
Hi,

I think you are absolutely right. There is a peak at 270nm seen on nanodrop. Is there any way I can clean up the samples with a kit. Will that help? What can be a good solution to this problem? Will I need to do the isolations again?

Thanks
I have this issue with Trizol sometimes. Two or three washes with 75% ethanol, rather than the 1 wash in the protocol, usually prevents it from happening. I also never to try to recover all of the aqueous phase after adding chloroform.

If you already have samples like this, precipitate the RNA and wash the pellet a few times with 75% EtOH.
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