Checking the optimal hybridization temperature as described above.In order to minimize the rain, you can optimize your digital PCR reaction by: Rain is clearly visible on the two first plot whereas plasmid digestion has removed most of the partitions corresponding to rain. 1D dot-plots of a digital PCR experiment in partition using one fluorescent assay targeting a non-digested plasmid (A and B) and a digested plasmid (C). Various factors may be at the origin of the “rain” such as, template degradation, PCR inhibitors, polymerase errors, primer dimer, target accessibility (Figure B) or non-homogenous distribution of fluorescence into partitions.įigure B. The rain makes the threshold setting harder, and thus may affect quantification. In these partitions, amplification/hybridization efficiency is sub-optimal. We call “rain” partitions that have intermediate fluorescence values between the positive and the negative populations. Presence of partitions of intermediate fluorescence (rain) If using a system where you can vary acquisition parameters, you may also try to tune the exposure timeģ.In this case, you may see a high fluorescence basal level, and, since there are only a few intact probes for PCR, a low signal for the positive partitions. Check whether your probes are too old! Depending on how long your probes have been stored, whether they have been stored properly, or whether they just encountered too many freeze-thaw cycles, it is possible that your probes are already hydrolyzed.Double-quenched probes will give you a lower basal fluorescence signal, and therefore a higher separability. Check the type of probes you are using.Check for the highest hybridization temperature that gives you an optimal separability and no rain (see below). These may be higher than the ones usually recommended in qPCR. Check manufacturers recommendations on primers and probe concentrations to be used in your digital PCR system.Here are a few tips to improve the separability of your populations: The fluorescence of the positive population (blue) is clearly separated from the negative population (dark grey), placing a threshold is therefore unambiguous. The positive partition population (blue) is barely separated from the negative population (dark grey), rendering thresholding difficult on this 1D dot plot. When you run an assay for the first time, you might not distinguish the negative and positive populations on a digital PCR system (Figure A).įigure A. Difference in fluorescence amplitude between negative and positive populations Template quality is also a factor that is important to monitor as base degradation such as cytosine deamination and 8-Oxo-2′-deoxyguanosine formation due to oxidative damage may lead to base transversion during PCR amplification.įor more information about DNA preparation, have a look at the “ DNA Preparation For Digital PCR” item. To limit this phenomenon, it is crucial to prevent DNA contamination in the laboratory or cross-contamination from well to well. If there is no evident source for DNA material to develop your assay, you may use synthetic oligos as templates for optimization.įalse positives are also a factor that contributes to the diminution of the sensitivity of the digital PCR assay. The DNA solution used should be devoid of contaminants and potential inhibitors and have good absorbance (A260/230 and A230/260) ratios. Ideally, the DNA template should be in the same form as it will be in the sample you will want to assay : sheared in small fragments if targeting circulating cell-free DNA or relatively intact if assaying genomic DNA.
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