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Old 08-01-2011, 11:40 PM   #1
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Default Help with smallRNA Truseq kit - Agilent results? (repost from Illumina forum)

Hi guys! I'm a newbie to this forum so please bear with me! I did ask in the Illumina forum yesterday but didn't get any replies, so I thought I'd ask the RNA experts..

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
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Old 08-02-2011, 04:58 AM   #2
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Location: Purdue University, West Lafayette, Indiana

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This is after PCR "enrichment"? If so, that is the multimeric or hetero-strand dimer product common in TruSeq libraries. It is called, variously, "bird nesting", "daisy chaining", "bubble products", etc. For my formulation of various possibilities, see:

The good news is that it usually is not a problem because most of the time the extra peak disappears after strand denaturation. You could try running a pico RNA chip on the DNA after denaturation. See:

But, should the sample strands re-anneal, you may get confusing results due to single stranded molecules running slower on Agilent chips than double stranded ones:

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Old 08-03-2011, 12:05 AM   #3
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Hi Phillip

Wow I hadn't realised that this was such a well-known problem! I hadn't checked the other forums as I assumed it was a problem restricted to miRNAseq, rather than a general Truseq issue. It definitely looks like it could be dimer product - it is roughly twice the size of the desired product (~270bp cf. 145-160bp).

Thanks very much
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