Hello all! I am working on a liquid biopsy. I have extracted cell-free DNA aka cfDNA from composite blood using the Qiagen kit. I have first isolated Plasma from blood and then performed cfDNA extraction. I want to detect the ratio of ctDNA/cfDNA in the sample. What experiments should I perform next?
Hi Ria,
Measuring the ctDNA content in a cfDNA sample is perhaps less straightforward than you would have hoped.
If you happen to know any mutations that are in the tumour, the easiest way is perhaps to do a digital PCR (dPCR) experiment targeting both the wildtype and mutant allele. This will give you a reasonable first estimate of the ctDNA/cfDNA ratio in your sample. This assumes that the mutation you picked is homogeneous in the tumour tissue.
More complex (and expensive) methodologies may be able to give more accurate estimates of the ctDNA content.
As an aside, since you seem to be fairly new in liquid biopsies, you might want to consider running a BioAnalyzer or TapeStation (or similar) assay to check the quality of your isolated cfDNA. If you see any high molecular weight (HMW) DNA, this may indicate that your plasma was contaminated with cellular DNA from the white blood cells. This will dilute the ctDNA signal in the dPCR and confound your results.
Good luck with the experiments!
Daan
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