Two cDNA Peaks on Bioanalyzer

[patkrat]

I used 200 ng total RNA input and the KAPA HyperPrep Kit to generate cDNA libraries. Usually, our libraries look like Sample 1 in the attached pdf, but this time we saw two peaks and a trailing 'belly'. Does anyone have an idea what this could be? Of note, for this High-Sensitivity Bioanalyzer run, I left the cDNA samples at room temperature for 30 min, together with the Bioanalyzer reagents; I usually leave the samples on ice, hence I mention it; maybe this could be a reason? Otherwise, I am thinking of overamplification, i.e. too many cycles for the library amplification step. It would be great to get some feedback and advice on this.

[pmiguel]

Hi patkrat,
Yes, the apparently longer products are likely "bubble products". It is hypothesized that bubble products form during PCR when the concentration of denatured products is high enough to allow strands from different library molecules to anneal at their adapters, leaving the two insert strands unannealed because they are not related. The floppy insert strands apparently retard the progress of these bubble product through the sieving matrix of the chip.

You can ignore the issue if you like. The bubble products will denature along with the normal double stranded products during denaturation and cluster normally. But if you need an average size for your library to calculate its concentration (eg, for qPCR), then you want to use the left peak to determine that.

--
Phillip

[patkrat]

Hi patkrat,
Yes, the apparently longer products are likely "bubble products". It is hypothesized that bubble products form during PCR when the concentration of denatured products is high enough to allow strands from different library molecules to anneal at their adapters, leaving the two insert strands unannealed because they are not related. The floppy insert strands apparently retard the progress of these bubble product through the sieving matrix of the chip.

You can ignore the issue if you like. The bubble products will denature along with the normal double stranded products during denaturation and cluster normally. But if you need an average size for your library to calculate its concentration (eg, for qPCR), then you want to use the left peak to determine that.

--
Phillip

Thanks for your response, Phillip. In fact, I ran another HighSens chip with 1:10 dilution of the cDNA library and found that this completely resolved the issue. While you may be right about the bubble product, I think that the main issue in this case actually was simple overloading of the chip (which has a 10 ng max capacity); I sometimes do a quick photospec to get the library concentration before running it on the Bioanalyzer, but this time the concentration was way off. I checked with the Qubit and found that I actually had up to 30 ng/ul libraries.

Conclusion: Overloading of the chip causes a split peak, but 30 ng/ul also indicates too many library amp cycles, which I will reduce from 13 to 12 (or maybe even 11) next time.

Thanks again for your answer.

[pmiguel]

Thanks for that follow-up. Interesting.

--
Phillip