Help with smallRNA Truseq kit - Agilent results?

[Tali7]

Hi guys! I'm a newbie to this forum so please bear with me!

I've prepared lots of Illumina libraries before (RADseq, RNAseq..) and am now doing miRNAseq using the TruSeq smallRNA kit, using total RNA input.

I've got to the pre-gel purification stage (post-PCR) and have run an aliquot on the Agilent Bioanalyser as encouraged to do by the protocol. However, the on-site Bioanalyser isn't new enough to use the suggested High Sensitivity DNA chips, and I had to use a normal DNA chip. So basically I didn't really know what I was looking for in my results graphs.

I do know that I am supposed to see a peak at ~145-160bp (the adapter-ligated miRNA size). And I do have small peaks in roughly that size region.

HOWEVER, I also have maaaassive peaks at ~270bp. I have no idea what this could be, and nobody I've spoken to knows either! I know it isn't contamination, because a sample that is effectively my -ve control (don't ask) didn't have any peaks. I'm just concerned that, as the peaks are so big, the adapters (although designed to ligate to miRNA) have also ligated to bigger RNA too, and so have also been amplified in PCR.

Would this be possible, and/or does anybody have any idea what these 270bp peaks are doing in my library prep?!

Thanks so much

[chaos81]

The most important thing is to have results in your target region. yes, you will get other ligated material, but after size selecting this should be primarily wiped out. As long as you see results in your target region then I would continue on with the protocol. and I use a DNA 1000 chip at that step and we have the capability of using a high sense chip. hope this helps.

[jpelletier]

I have seen the same thing in my miRNA library prep too! I was told that it is probably adapter-ligated t-RNAs. They were indeed effectively removed by the gel size-selection.
cheers!

[mnkyboy]

It is mainly tRNA. Run the BA again after you gel select.

[mboth]

I have done a few TruSeq small RNA library preps. You are adding ca 120bp of adapters/primers to your fragments, so the tRNA peak is at 180 - 190bp, not at 270. I suspect that 270bp represents rRNA. I also had a massive peak there, but after ribodepletion it was much smaller. Hope this helps

[pmiguel]

Or this is the common "bubble product/daisy chain" issue seen with TruSeq libraries. If so, you libraries are fine. Try denaturing them and running them on an RNA chip -- this will remove artifacts caused by ectopic reannealing of adapters. Here is what we do sometimes:

http://seqanswers.com/forums/showthread.php?t=12523

--
Phillip