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Old 11-19-2015, 07:25 AM   #1
Holioneok
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Default Inconsistensies in RINs between Bioanalyzer and Tapestation

Hi everyone,

I've recently isolated RNA using Qiagen kit (only obtaining RNA >200 nts) and ran the samples on High Sensitivity Screentape. The RINs are quite good, between 7.5 and 8.5, and 28S peaks higher than 18S peaks. There seems to be some smearing so quality could be a bit better but the results indicate the quality is decent.

Next I sent these samples to a sequencing facility. Unfortunately the package got stuck at customs and was underway for about 55 hours, much longer than expected. Nonetheless the people at the facility informed me that the samples were still frozen and on dry ice when they opened the package so I thought I was safe.

However their QC, using a BioAnalyzer showed much different results than mine, with RINs dropping to 5.3 (where I measured them at 8). When I rerun the samples on the Tapestation I get similar results as before, so still decent quality (RINs around 8).
The people at the facility suggest it might be because of the different machines since the samples were frozen when they arrived so likely nothing bad happened during transit.

However when I compare the electropherograms from TS and BA they look different (this is also true for the gel pictures), which is not something I would expect to arise from a difference in hardware since, as I understand it, the peaks in the electropherogram are what the RIN is based on. I would attribute such to degradation somewhere along the way but my experience with RNA is not extensive.

Has anyone here come across a difference like this? Could it indeed be because of the different machines? Or could the samples have degraded during transit after all? Many thanks for your input!

Here's a picture with snapshots from both ScreenTape and Bioanalyzer

Last edited by Holioneok; 11-19-2015 at 07:35 AM.
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Old 11-20-2015, 07:53 AM   #2
NextGenSeq
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Looks like real degradation to me.
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Old 11-20-2015, 08:23 AM   #3
blancha
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Looks degraded to me too.

Never take a chance on sequencing RNA with potentially low RIN.
It's a nightmare to analyze afterwards, and will leave a lot of people bitter and unhappy from having wasted time and money trying to analyze degraded RNA.
I'm talking from experience.
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Old 11-20-2015, 02:38 PM   #4
adam.geber
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I agree that your sample looks like it has degraded but this wouldn't be the first time that I've seen a surprising amount of variation between samples run in parallel on the Bioanalyzer and Tapestation. Additionally, I've frequently seen an odd BioA electropherogram trace on RNA samples from column cleanups of phenol/chloroform extracts: basically, the 28S peak is dampened relative to the 16S (~1:2 28S:16S) but purification returns the proportions to 1:1 or closer to the 2:1 ratio of a perfect RIN score. See here for a paper describing this exact phenomenon. What kit were you using for your extractions? I haven't seen this 28S dampening on the Tapestation so far but I haven't had the chance to check my older samples on it for a comparison.

Alternatively, it could be that your sequencing facility needs to do a RNAse decontamination of their BioA pins, although if it's a reputable place then this is likely not the case.

Also, depending on what you're sequencing the degradation may simply be an unavoidable consequence of freeze-thaw cycles. What will be done with your sample at the facility?
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Old 11-20-2015, 11:17 PM   #5
Holioneok
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Thank you all for your input!

Quote:
Originally Posted by blanca
Never take a chance on sequencing RNA with potentially low RIN.
It's a nightmare to analyze afterwards, and will leave a lot of people bitter and unhappy from having wasted time and money trying to analyze degraded RNA.
Absolutely, it's not my intention to continue with the low RIN samples. But before sending new samples I will have to figure out first what the cause of the low RINs on their end is.

Quote:
Originally Posted by adam.gember
I agree that your sample looks like it has degraded but this wouldn't be the first time that I've seen a surprising amount of variation between samples run in parallel on the Bioanalyzer and Tapestation. Additionally, I've frequently seen an odd BioA electropherogram trace on RNA samples from column cleanups of phenol/chloroform extracts: basically, the 28S peak is dampened relative to the 16S (~1:2 28S:16S) but purification returns the proportions to 1:1 or closer to the 2:1 ratio of a perfect RIN score. See here for a paper describing this exact phenomenon. What kit were you using for your extractions? I haven't seen this 28S dampening on the Tapestation so far but I haven't had the chance to check my older samples on it for a comparison.
Thanks for the paper. I use the Qiagen kit for extraction, then DNAse treatment and then another column cleanup for purification and concentration of the sample. Given the extra cleanup I doubt Trizol contamination would be the cause.

Quote:
Alternatively, it could be that your sequencing facility needs to do a RNAse decontamination of their BioA pins, although if it's a reputable place then this is likely not the case.
Could be, but it's a reputable place and other samples were run together with mine (including some control RNA) and they looked fine.

Quote:
Also, depending on what you're sequencing the degradation may simply be an unavoidable consequence of freeze-thaw cycles. What will be done with your sample at the facility?
As far as I know, samples aren't unnecessarily thawed and frozen. I think for their QC they thaw the samples and quantify using Qubit and at the same time run them on BioAnalyzer.
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Old 12-01-2015, 11:38 AM   #6
DarkQ
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Your samples seem to contain gDNA contamination based on the first diagram. The "shoulder" region beneath 28S/18S are gDNA according to agilent.
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Old 12-01-2015, 01:59 PM   #7
adam.geber
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DarkQ, wouldn't we expect to see more high-molecular weight fragments in the Tapestation trace if there were truly contaminating gDNA? I've seen the "shoulder" in samples that have received double DNase treatments (both on-column during the extraction and in-solution before a cleanup) and had written it off as an artifact of Agilent's curve-fitting algorithm.
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Old 12-02-2015, 03:13 PM   #8
kerplunk412
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In my experience degraded RNA shows a strong peak around 100 nt on the Bioanalyzer, so that doesn't exactly look like degraded RNA to me. Since your purification removed the <200nt RNAs, the ~100nt peak would be even more obvious if the RNA was degraded. The 18S peak seams just as strong in both traces, which also makes me think this may indeed be a case of the 28S peak being masked and not RNA degradation.

Depending on the capabilities of your facility and the quantity of RNA you sent, you could have them run it on an agarose gel to check the quality. A denaturing gel isn't necessary to just check the 18S/28S bands.

RNA isn't quite as sensitive as many think, as long as it isn't nuclease contaminated, so I also doubt an extra freeze-thaw or two is causing what you are seeing.

Edit: If that was a Nano chip they ran your samples on you could have them try running it on a Pico chip, or vice-versa.

Last edited by kerplunk412; 12-02-2015 at 03:15 PM.
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Old 12-02-2015, 06:11 PM   #9
DarkQ
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I agree that gDNA should have much higher molecular weight. It was based on a poster/publication from Agilent where they saw similar things and noticed the shoulder was DNase sensitive. Also when they manually added gDNA into the RNA sample the shoulder appears. My guess would be some gDNA would be sheared during tissue processing or RNA purification steps and those were the ones showed up in Bioanalyzer.
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