QIAamp DSP Circulating NA Kit (50)

From QIAGEN, this silica-membrane-based system is designed for the isolation and purification of circulating cell-free DNA and RNA from human blood plasma samples for in vitro diagnostic use. Intended for professional users such as technicians and physicians trained in molecular biological techniques, it is manufactured in accordance with GMP requirements. The procedure omits phenol-chloroform extractions; nucleic acids bind specifically to QIAamp Mini columns while contaminants and PCR inhibitors are removed during the wash steps, yielding purified nucleic acids for elution.

Performance was evaluated across three blood collection tubes — BD Vacutainer K2EDTA, PAXgene Blood ccfDNA, and Streck Cell-Free DNA BCT — with nucleic acids from 1 ml plasma inputs successfully applied in qPCR, digital droplet PCR, and RT-qPCR workflows. Total DNA yield from 5 ml plasma input ranged between 50–150 ng when measured with the Qubit HS dsDNA assay; in NGS analysis, >98% of generated reads mapped to the human genome and >99.8% of positions in the regions of interest had a base coverage of ≥500x. Precision testing using 144 purifications across three operators, three days, three instruments, and three kit lots gave a total SD of 3,120 copies/ml and total CV of 12.05% (mean 25,894 copies/ml). A linear increase in DNA yield was observed across 1–5 ml plasma input volumes. Eluate stability for DNA extended up to 12 months at −30 to −15°C or −90 to −65°C, and up to 48 hours at 2–8°C or room temperature (15–25°C); for RNA, up to 6 months at −30 to −15°C or −90 to −65°C, and up to 48 hours at 2–8°C. No interference was observed with hemoglobin, triglycerides, EDTA, caffeine, albumin, conjugated bilirubin, or unconjugated bilirubin when qPCR was used downstream. A cross-contamination study in which 105 copies of HBV virus were spiked into 5 ml or 2 ml plasma showed no cross-contamination within the system.

Two protocols, Classic and Breeze, both process up to 5 ml plasma in 1 ml steps on QIAamp Mini columns via a vacuum manifold in four steps (lyse, bind, wash, elute), with flexible elution volumes between 20 µl and 150 µl; the Breeze protocol is optimized for reduced hands-on and turnaround time. Isolated nucleic acids are suitable for downstream applications including NGS, RT-PCR, digital PCR, multiplex PCR, and quantitative PCR. Because free-circulating nucleic acids in human plasma typically range from 1–100 ng/ml and may be fragmented (DNA <1000 bp, RNA <1000 nt, miRNAs as small as 20 nt), fluorometric methods such as RiboGreen for RNA and PicoGreen for DNA are recommended for quantification.