I did a RNA isolation with a Polysome Pulldown + TriZol, followed by a purification with a RNeasy Kit. With the isolated RNA a transcriptome should be generated.
My samples on the Nanodrop looked quite clean.
(Exemplary sample value for 260/280 is 2,19 and for 260/230 is 2,48. I know that the 260/230 is a bit high but I couldn't find an explanation why. Also it shouldn't be a problem for generating the transcriptome I was told.)
Now I got the Quality control data, which were measured with a Agilent 2100 Bioanalyzer and they look quite strange.
18s/28s rRNA bands cannot be clearly descriminated, although the sample itself doesn't look degraded. You don't see a smear but clear distinct peaks. See attached an example.
Does anyone has an explanation for this?
Also an additional note: If I digest my sample with RNases, I cannot find any bands afterwards. So it seems those bands are RNA and not DNA contamination or whatsoever.
Don't know if it is important but those are c.elegans samples.
Also the all the sample look quite the same (n=6). You can't see a big difference between days.
Has someone experienced this before or does know what happened in my samples?
I am happy about every help I can get. :)
Hi Philip,
Few quires, what did you use as elution solution for your RNA, you can first check the integrity(quality) of your RNA by 2% agarose gel(RNase free condition), then go for the nanodrop,qubit or agilent bioanalyzer for quantity.
After quality, if still you got the same trend then ,either your RNA is degraded or yield is very low.
Hi Abhijit,
I used RNase free water (not DEPC treated) for elution.
Sadly I didn't do a gel for those 6 samples but for a different sample obtained the same way. I'll attach it to this answer.
Nanodrop values and plot looked good. The values were all in the range as stated in my first post.
Gel pic look good, did you get same bioanalyzer pics(as mentioned earlier) for these samples also.
Generally I also got some issues with bioanalyzer, but after lots of experiment on it was found that due to unusual eluetion buffer,it gives wrong pics.
In your case you can use (after agarose gel),directly qubit for quantification or else you can just run only elutent (without RNA) in a single well in bioanalyzer, to check the effect of elution buffer.
Best of luck.
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