Tapestation profile weird

[mark_6060]

Hi all,

We are outsourcing RNA-seq along with RNA isolation from a gram positive bacterium. The service provider has sent the QC report (Tapestation) for the isolated RNA.
The first sample is said to have failed and has the following profile.


For other sample, the RNA isolation is being claimed to have succeeded with the following profiles.






Do you have any suggestions regarding the following points?
(1) Why there is weird peak between 25 and 200 and also a corresponding darkest band in all the samples?
(2) Shall the service provider be asked to proceed with the library preperation for the bottom 3 samples?

Many thanks

[Ben3]

mark_6060 it's been a while since I've done any bacterial work or run that Tapestation, but to me, it looks like the RNA is a bit degraded and that's why there are large peaks at the end.

Do you have any older Tapestation runs to compare this to? Also, I remembered that for RNA Tapestation runs, the RNA has to be put into a thermocycler for denaturation prior to running on the screen tape. If the temperature is wrong it can degrade the RNA or cause it to run strangely on the screen tape.

Depending on your application, slightly degraded RNA might not be a huge deal. But first I would look at other runs and see how they compare first.

[mark_6060]

Ben3, thanks for your reply. These samples were analyzed for the first time; so I don;t have their earlier profiles. However, I do have profiles (pasted below) for some other samples that have passed the QC on the current batch.

[Ben3]

mark_6060 those profiles look much better. That's more of what I expect out of quality RNA. Seeing how they compare, I would say the RNA from the first charts is slightly degraded, but not that bad. I think the question would be how comfortable you would feel if there was some slight degradation.

Depending on the library prep kit and the aim of the study, it might not matter too much if there's a small bit of degradation.

[AngelaT]

I realize this post is older, but I just had what seems like the same problem, so I figured I'd comment anyway.

I'm not familiar with RNA, so I can't comment on the sample graphs themselves.

I have had samples fail QC, even though they had very large peaks. What was happening is that the sample concentration was so high that it was stronger than the ladder, so the software was not able to pick up the ladder peaks. Try diluting the samples a bit and trying them again.