Circulating cell free DNA (ccf-DNA) was extracted from 200 μl of plasma samples using the NucleoSpin® Plasma kit (Machery & Nagel), following manufacturer’s instructions. The circulating cell free-mtDNA (ccf-mtDNA) and cell free-nuclear DNA (ccf-nDNA) were quantified by droplet digital-PCR (dd-PCR, Bio-Rad) with Taqman-based methods, amplifying MT-ND2 and FASLG [22 (link)] and MT-ND1 [23 (link)]. The ccf-mtDNA and ccf-nDNA were expressed as copies of target gene on μl of template analyzed (copies/μl template).
Nucleospin plasma kit
Manufactured by Macherey-Nagel
The NucleoSpin® Plasma kit is a product designed for the isolation and purification of viral and bacterial nucleic acids from plasma or serum samples. The kit utilizes a silica-based membrane technology to efficiently capture and purify the target nucleic acids, providing a reliable and reproducible method for downstream applications.
Automatically generated - may contain errors
2 protocols using nucleospin plasma kit
1
Quantifying Circulating Cell-Free DNA
2
Quantification of Circulating Cell-Free DNA
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Serial blood samples (250 μL) were collected by retroorbital bleeding from PDX mice using microtainer tubes with dipotassium EDTA (BD Biosciences, San Jose, CA). cfDNA was isolated from 100 µL of the plasma samples using the NucleoSpin Plasma kit (Macherey-Nagel, Düren, Germany). cfDNA concentration in the plasma was subsequently determined by quantitative polymerase chain reaction using the Custom TaqMan assay with the 7900HT Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). Human β-actin primer pair and probe set are as follows: forward primer, 5′-ATCCTAAAAGCCACCCCACT-3′; reverse primer, 5′-CTCAAGTTGGGGGACAAAAA-3′; and probe, 5′-FAM-CACAGGGGAGGTGATAGCAT-MGB-3′. Serial dilutions of genomic DNA extracted from the Hut78 CTCL cell lines were used to calibrate for cfDNA quantification. Sample cfDNA concentrations were extrapolated from the standard curve using Prism software v.6.0 (GraphPad Software, La Jolla, CA).
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