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Old 07-03-2014, 12:25 PM   #1
Francy87
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Location: Italy

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Unhappy mRNA traces on Bioanalyzer

I know it's a discussed argument, but I'd like to have an opinion about my Bioanalyzer traces.
I extracted total bacterial RNA from food samples, then I used the Bacteria RiboZero kit to purify from rRNA. I don't know how my total RNA looked like before the RiboZero depletion, since I didn't run it on the BioAnalyzer, but after the rRNA depletion it looked like in the BA traces attached.

The sequencing core said I can't go ahead with the library preparation… But I saw on the RiboZero website that after the purification, I should have only a peak at 100-140nt, that is what I have. I can't understand why my traces are different from what epicenter write on the website.
Any comments about this? Does anyone has ever obtained libraries from this kind of mRNA?
Attached Files
File Type: pdf mRNAassay_ PicoKit_rRNA_depleted_2014-07-02.pdf (2.24 MB, 105 views)
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Old 09-07-2014, 04:27 PM   #2
littlefairy
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Hi,
Sorry I can not answer your questions
Could you please tell me how to print the xad file to pdf like what you did.
Thanks
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Old 09-07-2014, 06:28 PM   #3
nucacidhunter
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Quote:
Originally Posted by littlefairy View Post
Hi,
Sorry I can not answer your questions
Could you please tell me how to print the xad file to pdf like what you did.
Thanks
On Agilent 2100 Expert software go to File>print which will open a print dialogue box. In the box under “Save To File” section tick PDF box.
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Old 09-08-2014, 07:18 AM   #4
littlefairy
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That works well.
Thanks a lot.
Hopefully, you will solve the problem.
By the way, my total prokaryotic RNA sample yesterday is of low RIN value, merely 6, which means RNA-seq could not be done on this samples.
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Old 09-08-2014, 07:40 AM   #5
pmiguel
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Quote:
Originally Posted by Francy87 View Post
I know it's a discussed argument, but I'd like to have an opinion about my Bioanalyzer traces.
I extracted total bacterial RNA from food samples, then I used the Bacteria RiboZero kit to purify from rRNA. I don't know how my total RNA looked like before the RiboZero depletion, since I didn't run it on the BioAnalyzer, but after the rRNA depletion it looked like in the BA traces attached.

The sequencing core said I can't go ahead with the library preparation… But I saw on the RiboZero website that after the purification, I should have only a peak at 100-140nt, that is what I have. I can't understand why my traces are different from what epicenter write on the website.
--
Phillip
Any comments about this? Does anyone has ever obtained libraries from this kind of mRNA?
Pico chips are very sensitive. So the lack of any visible mRNA makes it seem like nearly all of your sample was lost and/or degraded during purification.
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Old 09-09-2014, 04:47 PM   #6
kerplunk412
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I agree that it looks like your RNA is degraded. It would be a good idea to check your samples on the BA (or by agarose gel) before starting the depletion to determine if the problem arose from your samples or the rRNA depletion process. It is always a good idea to assess the quality of your starting material before starting purification and library prep.

If these are very precious samples you might still be able to make libraries from them, but you should probably skip fragmentation and possibly modify bead cleanup steps to retain smaller products.
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