Are you looking to start using blood samples in your translational research? Maybe you’re interested in metastatic breast cancer, or non-small cell lung. Or maybe the challenges and limitations of solid tumor samples have you thinking… it’s time.
Let’s take a closer look at DNA yield, and the reality of working with cell-free DNA from plasma. Cell-free DNA (or cfDNA) refers to all non-encapsulated DNA in the blood stream. A portion of that cell-free DNA originates from a tumor clone and is called circulating tumor DNA (or ctDNA). cfDNA are nucleic acid fragments that enter the bloodstream during apoptosis or necrosis. Normally, these fragments are cleaned up by macrophages, but we believe the overproduction of cells in cancer leaves more of the cfDNA behind. These fragments average around 170 bases in length, have a half-life of about two hours, and are present in both early and late stage disease in many common tumors including non-small cell lung and breast. That said, cfDNA concentration varies greatly, occurring at between 1 and 100,000 fragments per millilitres of plasma.
If you’ve been in the next-generation sequencing space for a while, you might want to know about sensitivity and specificity. We demonstrated high sensitivity and specificity for variants at frequency greater than point one percent with 20 nanograms input DNA, and greater than point five percent with 5 nanograms . To see more on how to do that, check out our video on cfDNA assays. You may have heard about using cfDNA for analyses at point zero one percent limit of detection. While the technology exists to reach .01% (digital droplet for example), there’s a biological limit we need to talk about.
From a 10 mL blood sample, using the Applied Biosystems MagMAX Cell-Free DNA Isolation Kit, we routinely obtain about 20 nanograms of cfDNA, or around 6 usable molecules for analysis. Some quick math shows us that at 1% limit of detection, we can expect around 25 molecules. At 0.1% only 2-3 molecules are expected to be present. That means below 0.1% there may not be any molecules present for analysis. Remember, too, that cfDNA concentrations are highly variable. They can be very low at critical stages such as early recurrence or development of resistance, where many researchers are now focused. To successfully isolate 20 nanograms of DNA from blood plasma, both manual and automated methods are available depending on your throughput needs.
DNA in hand, your research dictates which path to take at this point. You might choose digital PCR for recurrence monitoring studies. For research in therapy selection, a next-generation sequencing assay may be preferred. Wherever your liquid biopsy research takes you, we can help you get there with complete solutions for cfDNA analysis from just a few targets to multi-gene assays.
I hope we’ve given you some things to think about as you consider using cell-free DNA in your lab. But I’m sure you’ll have some questions. So submit your questions at thermofisher.com/ask and subscribe to our channel to see more videos like this. And remember, when in doubt, just Seq It Out!