Bioanalyzer RNA QC

[cahe]

Hi everyone

I am ready to start my first library prep with the TruSeq v2 kit, but already got stopped after a Bioanalyzer Pico run to verify the quality of my extracted RNAs. The RIN is N/A, undetermined because of a baseline drop or increase. I was so far not able to find such a profile in troubleshooting examples and am hoping that someone here can help figuring out what is going on with some of my samples. Unfortunately, I do not have access to the instrument and software. Samples were extracted from C. elegans using Qiazol followed by RNeasy mini, Bioanalyzer file is attached (Please do not consider 23 and 24).

Thanks a lot
Caro

[DRYTCYV]

Hi,

First of all, Ladder peaks size are not adjusted.
Sample 3 need to be adjusted too.
The profile looks ok, but a strange baseline (negative values).

This is my quickest answer

Cheers

[pmiguel]

Yes, the person that ran your bioanalyzer chip needs to force the software to recognize the correct position of the 25 base marker in the molecular weight marker. Same thing for sample Ev-III, the dumbo software mis-called the 25 base marker peak. It needs to be forced to recognize the right peak.

My guess is that your samples are fine. Pico chips are the worst for irregularities. But they are very sensitive...

If you poke around on the Agilent site, you can find the Expert 2000 software available for download. It is kind of obnoxious -- has some weird licensing modules that stay resident on the computer you infect with this package. But the software itself is very powerful. If you had it, you could just ask your agilent facility for the .xad file and make the adjustments yourself.

Myself, I feel like I have both arms tied behind my back just looking at the .pdf.
--
Phillip

[cahe]

Hi
Thank you for your comments. Seems like a rather bad service from a core facility that they don't even make adjustments to the software.
They provided me with the .xad files, so I will try to get the software.

Thanks
Caro

[kerplunk412]

I guess you have probably solved your problem by now, but I thought I would comment anyway in case it can help anyone else. Although you didn't get any RIN values from the BA, the 28S and 18S peaks in your samples are sharp and the 28S is roughly twice the intensity of the 18S, which indicates very high quality RNA that should work well for library prep. For comparison, sample 23 in the pdf is highly degraded and sample 24 is somewhat degraded but still probably okay for library prep. I have run a decent amount of RNA on the Bioanalyzer, and I am confident that all of your samples would have a RIN of >9.