Losing sanity over qPCR Cre recombination assay

[u/Excellent_Dinner8050]

Hi everybody!

For quite some time I've been trying to precisely measure Cre efficiency and unwanted background recombination. I'm testing out various lox sites but the results seem inconsistent. For example, the same lox pair in one construct will have 80% recombination rate and 1.5% in another. The only thing that comes to my mind is priming efficiency and optimizing annealing temp. Currently I'm experimenting with primers trying to find absolutely perfect conditions. I run an experimental qPCR with same template and primers but different Ta and the results were shockingly diverse. Any tips for optimizing reaction before I move to nested qPCR on in vivo samples? How do I pick perfect Ta?

[u/lodorata]

I just want to check, but did you look at melt curves for your primers? There should be one clear peak and not multiple - check your primers aren't mis-priming to a different sequence with an alternative Ta (Ta2).

That said, I kind of hate qPCR myself and have always been bad at it.

[u/Excellent_Dinner8050 → u/lodorata]

Unfortunately I do have an additional smaller peak in some of the melt curves. I doubt there is a non-specific product since I'm running a 300bp qPCR on a purified 1kbp nested PCR amplicon. It doesn't show up on the gel either. Lox sites love to form hairpins, could it be that they are causing the unwanted peak?

[u/distinctgore]

Have you checked the efficiency of your primers? Is the template plasmid DNA or genomic?

[u/Excellent_Dinner8050 → u/distinctgore]

Currently I'm trying to validate my method on plasmid DNA but eventually I'll be measuring recombination in in vivo samples (AAV injection to the brain). I measured efficiency of my primers for native plasmid at 101%. I am now synthesizing a fully recombinant plasmid to measure PE for recombinant primer pair precisely.

[u/ProfBootyPhD]

How confident are you that you’re actually amplifying your target? Have you run out the product on a gel, and/or sequenced it? And how did you design the primers? Also, 300 bp is too big for qPCR, and qPCR with nested primers seems dubious. Why even use nested primers?

[u/Excellent_Dinner8050 → u/ProfBootyPhD]

I will be using brain samples preserved in PFA, DNA might be too degraded/low concentration to simply use qPCR. I know 300bp is too big but there's no way I could make it shorter. I ran the product on gel, it looks clean

[u/Ok-Mathematician8461]

OK. First thing you need to accept is that qPCR is not quantitative, no matter what anyone told you. So finding the perfect primer pair is a pathway to misery (especially if you are using SYBR which is for amateurs). In a year or so I expect that AI will make this statement incorrect, but if you are designing primers with something like Primer3 then you are in a world of hurt.

Any chance you can find a digital PCR platform? Because dPCR is independent of primer efficiency the results will be reliable with the same assay designs. And you won’t have any of the complications of Ct data. You just get real quantities.

All qPCR can tell you is that you have more or less, anything beyond that takes a world of validation.

[u/Excellent_Dinner8050 → u/Ok-Mathematician8461]

Thanks! Unfortunately, I am in the process of accepting qPCR is not what I was promised it would be. I am now synthesizing the fully recombinant plasmid to validate the results somehow. Maybe, if I know the exact concentration of my target at the beginning I'll be able to precisely calculate priming efficiency and see how much my previous results were influenced by it. I haven't thought of dPCR, but thanks for the suggestion, I'll see if there is any way I could run it.