2100 expert_Eukaryote Total RNA Nano_S.O.S Strange results!

[ValeD]

Hi all! I'm really new here...and even with Agilent RNA 6000 Nano Assay Kit Now I'm testing some samples to prepare them for the core facility which will'do library (mRNA and miRNA) for HisSeq Illumina. The first assay (see attached), on RNA from rat muscle tissue previously extracted by Trizol, was almost good: RIN just below 8 and a little peak (and slight band in the gel) between 25 and 30 [s]. But when I re-did the extraction and bioanalysis (like 5 times till now ) maintaining the same conditions, I had bad results with this third peak growing in intensity and RIN reducing highly...the strange thing is that samples at nanodrop show good features, but not in the bioanalysis...I even tried to purify RNA (with NaAc) but improvement only on the nanodrop...I wonder what could be the reason of this peak and this very low RIN (around 2.8 now)...may be some contaminations?
Can anyone help me in this tricky, because I really don't know...
Thanks a lot in advance
V

[durandg]

Hi,

Would you have a pdf of your "bad" profile's Bioanalyzer runs? It could help to picture your problem and try to help.

Otherwise Agilent Techsupport or FAS can be a good help; it was the case for me in the past when i faced strange profiles sometimes...

D.

[ValeD]

2100 Agilent RNA 6000 Nano Assay Kit_ strange results

Hi durandg,
thanks for your kind reply!
here are attached the 'bad' profiles. in both assays the same samples were evaluated (well 11 and 12 are empty): in file n1 they were diluted 1:2, as all but sample 1 had conc over 500ng/ul, in file n2 I used them not diluted. strangely conc seems not influence the result, even if the Nanokit range is 25-500ng/ul (you know, actually I'm wondering even if the instrument is working properly ). anyway my problem is that peak/band between 25 and 30 [s] that surely interferes with miRNA library contruction...
V

[Lanlan]

similar results also here

http://seqanswers.com/forums/showthread.php?t=19367

[ValeD]

Hi Lanlan,
thanks! according to these considerations, if the band is due to all miRNAs you should have seen that before in other bioanalysis, right? so, is it more likely other contamination, like DNA one?
V

[durandg]

Hi ValeD,

Thanks Lalan for the link to the other thread! On my point of view, this is miRNA (probalby not degraded RNA because in that case it won't be a "huge" pack of degraded RNA only from that size but more randomly sizes).

I think the fact that your peak 18S and 28S "disappear" is due to the huge mass of this miRNA; the range of the kit is up to 500ng/ul but you have so much material in that miRNA that you're probably out of the range for the non-diluted chip. This can explain the fact that your peaks of interest are lowered and non visible in some cases.
I invite you to respect the range of the kit; for my own experience with this machine and this kit, i consider the data right up to 600ng/ul, above it i rerun the samples, once diluted. A true fact is that when a sample is out of range, the data that you obtain are wrong because of non linear (e.g. if the software shows you a sample at 800ng/ul and you dilute it 2x for a next chip, you won't obtain 400ng/ul. But this last data will be the correct one.
A last thing about the amount of material is that a too big amount can interfer with other wells as you one common "sheet" of gel spread in all the wells; if you have a too big amount of RNA that cause problem in a well for the migration, it can do the same effect to surrounding wells (problem when the case occurs close to the ladder). But these things are quite rare and honestly you have to load a loooooooot of material to have that.

So, if i were you, i would purify the samples to get rid of the miRNAs and then run it again on the Bioanalyzer -that i think yours work really well if i consider the ladder and empty wells migrations and the standard curve. ;-)

Good luck, enjoy this good machine!
D.

By the way, you can choose the number of wells to migrate in a run if your chip is not full (starting from well nb 1 of course). It can save time!

[ValeD]

Hi durandg,
many thanks for your experienced and useful suggestions!! I'll put them in practice and keep you informed about results.

ps: great! your good opinion about machine reassured me a lot

cheers
V

[durandg]

Hi ValeD,

Looking forward from your news with next results! I hope it'll help and you'll get rid of your problems.

Don't hesitate if i can help.
Cheers,

D.

[rnal]

Hi,
I think you can run a general agarose gel. I also get different results from 2100 for the same sample.
if the gel is good, I think you can trust you first 2100 result(RIN about 7).
and I often dilute the RNA in 100-200ng/ul(NanoDrop 1000) then run 1ul in 2100 RNA Nano 6000 and get good results.