Strange double-peak in Agilent RNA profile

[kgoglin]

We are learning to pay attention to extraneous peaks seen in RNA profiles from the Agilent RNA nano chip. If we see any sort of tiny peaks around the 23S peak, we automatically assume DNA contamination and repeat a second DNAse treatment. This seems to clear most of those tiny peaks and samples behave much better in downstream processing.

The last set of samples we extracted RNA from had to go through this second round of DNAse treatment but still a strange "double-peak" remained just before the 23S peak. Has anyone seen something like this before?

These particular RNAs were extracted with Trizol and glass beads were added to break up the cells. Perhaps the glass beads caused this somehow?

[buthercup_ch]

Sorry but this amples are supposed to be total RNA, right?
Then why observing 2 peaks is strange? you should observe several peaks actually, mainly those for 16S and 23S if working with bacteria (as I assume you are doing), additionally another peak for 5S and small rRNAs and tRNAs and in some cases even an additional low intense peak higher at high molecular weight.
How is that possible that the peaks, that show the presence of undegraded RNA will disappear by treatment with DNAse?

[kgoglin]

The two peaks I'm referring to are not the 16S and 23S. If you open the pdf file and look at the 30 hour samples, you will see what I am referring to.

We processed these samples through Ribo-Zero (for rRNA depletion) and the doublet-peak disappeared. These samples went on to make nice RNASeq libraries.

Thanks for you input.