Dissociation analysis for RNAseq library

[sehrrot]

Dear all,

I've been using qPCR assay with dissociation analysis to check primer dimers or other undesired fragments. I found bird-nesting peak (red plot) at higher temperature and am wondering what this peak means. (c.f. one single peak in the red plot is the one we expected as library)

[pmiguel]

Dear all,

I've been using qPCR assay with dissociation analysis to check primer dimers or other undesired fragments. I found bird-nesting peak (red plot) at higher temperature

I don't understand. The red peak overlaps the lower temp green peak. When you say "higher temperature" in this context, what do you mean?

and am wondering what this peak means. (c.f. green plot is the one we expected)

I see a "shoulder" red peak at 84 oC. Is that what you are looking for?

I have not done much with melting curves. What temp would one expect >100 bp dsDNA to melt at? I would have thought <90 oC.

--
Phillip

[sehrrot]

Dear Phillip

The peak around 79-80C indicates library. The one I am looking for is the peak (shoulder one) at 84C in the green peak. I just guess this is the fragment with high GC

[pmiguel]

Interesting. Do you have an example of one with a high amount of primer dimers?

--
Phillip

[sehrrot]

This is the first RNAseq samples I got. Before qPCR, I found primer dimer peaks at the bioA plot so repeated beads purification using 0.7:1 ratio (beadsNA library). Then I got primer-dimer-free library and supposed that there would be a single peak in dissociation curve analysis. But there's shoulder peak....

[pmiguel]

So you think that the higher melting temp peak is the primer dimer?
Can you use another assay to confirm? Like running an RNA Agilent chip on the library after strand denaturation? (95 oC for 2 minutes followed by snap cool on ice until the sample is loaded.) If you run the library strand-denatured, the short amplicons (primer dimers) won't be able to "hide" by annealing to longer library molecules.

--
Phillip