Hello everyone,
I'm in the initial stages of conducting a study involving the ddPCR (Bio-Rad QX200, QX600) and I ran into some problems involving recovery % calculations using direct quantification. I'm spiking a nucleic acid that essentially ends up being detected (and amplified) as a 100bp ssDNA template, and I get a concentration reading from the ddPCR of about 800 copies/uL. My issue is that by calculation, with 2.5pM final spiking concentration, I should get about 3 x 10^7 copies per 20 uL. This is of course not reflected in the results and above the actual detection capabilities of the instrument. Further dilutions down to 500 fM still show this issue. In most of the papers I've read, initial copies spiked in are determined empirically. Am I missing something in my calculations or is determining copy number by weight before spiking the only way? Thanks in advance!
Hi,
The maximum amount of copies that can be quantified per well is about 10^5, so you will have to dilute at least 300x more. That said, calculations based on weight can be off a lot.
I would start with a pilot experiment where you run a dilution series of your spike-in to know how much you actually want to add to your reaction. I would also not go for 100K copies, but take an amount that leaves room for flexibility. Based on that dilution series, you can then calculate how much DNA you would like to add to your samples. (This will depend of the elution volume and how much volume you will add to the ddPCR reaction.)
So, briefly, I would use ddPCR to calculate how much DNA you do have to start with.
In the next experiment, you can then calculate recoveries: run a ddPCR reaction including a spike-in control condition on the same plate (same amount as at 100% recovery) as your samples.
For example, if you spike in 10µL (previously diluted DNA) prior to extraction and elute in 100 µL --> At 100% recovery, you will end up with 0.1 µL spike in / µL elute (or a 1/10 dilution of what you initially added).
This concentration is also the concentration that you should add to your spike-in control reaction to calculate recovery.
Imagine you add 10 µL of sample to your ddPCR reaction then
--> your actual samples at 100% recovery will contain 1µL of spike-in in those 10 µL (0.1 x 10)
--> To your spike-in control reaction you can then add 10 µL of a 1:10 dilution spike-in (1µL spike-in + 9 µL water/carrier)
And then you can calculate the %recovery based on the copies/µL that you will obtain from the software.
% Recovery =( "spike in sample" / "spike in control") x100
Hi Caroline,
Thanks for your answer! It turns out I was on the wrong track focusing on recovery percentage but didn't realize (I'm picking up this study part way) that the initial protocol uses a (I think low efficiency) hybridization technique for miRNA that essentially un-directs the quantification. My next step is to set up a standard curve then see where to go from there. Thanks again!
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