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A Comparison of Cell-Free DNA Isolation Kits: Isolation and Quantification of Cell-Free DNA in Plasma.

Author(s): Sorber L, Zwaenepoel K, Deschoolmeester V, Roeyen G, Lardon F, Rolfo C, Pauwels P

Publication: J Mol Diagn, 2017, Vol. 19, Page 162-168

PubMed ID: 27865784 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of cfDNA isolation kit choice on total and KRAS-mutated cfDNA yield and cfDNA integrity. Plasma was obtained from two patients with benign pancreatic cysts and seven patients with pancreatic ductal adenocarcinoma (PDAC)(3 who underwent surgery and 4 with metastasized disease). A second specimen was obtained from one of the patients with PDAC with metastasis. No details of blood processing were provided. cfDNA was extracted from plasma using the EpiQuick Circulating Cell-Free DNA Isolation Kit, the first and second versions of the NEXTprep-Mag ccfDNA Isolation Kit, the PME free-circulating DNA Extraction Kit, the QIAamp circulating nucleic acid kit, and the Maxwell RSC cfDNA Plasma Kit.  DNA was quantified using digital droplet PCR and the KRAS Screening Multiplex Kit. DNA integrity was confirmed by real-time PCR of 105 and 236 bp fragments of GAPDH. The KRAS mutational status had been determined previously using DNA extracted from blood (all 10 specimens) using QIAamp circulating nucleic acid kit and from FFPE tissue using Cobas DNA Sample Preparation Kit (5 specimens with KRAS wildtype determined using blood).

   Summary of Findings:

   Compared to the QIAamp circulating nucleic acid, a comparable amount of total cfDNA was isolated using the Maxwell RSC cfDNA Plasma Kit (62.57 copies/µL versus 56.49 copies/µL with QIAamp) and significantly less cfDNA was isolated using the PME free-circulating DNA Extraction Kit (4.06 copies/µL, P<0.0005) or the second version of the NEXTprep-Mag ccfDNA Isolation Kit (6.08 copies/µL, P=0.001). Further, the yield was below the limit of detection for digital droplet PCR for three of 10 specimens isolated using the PME free-circulating DNA Extraction Kit and all 10 specimens isolated using the first version of the NEXTprep-Mag ccfDNA Isolation Kit or the EpiQuick Circulating Cell-Free DNA Isolation Kit. The authors attribute lack of digital droplet PCR products using the first version of the NEXTprep-Mag ccfDNA Isolation Kit to ddPCR inhibiting agents as real-time PCR cycle threshold values were comparable between versions of the NEXTprep-Mag ccfDNA Isolation Kit. While KRAS-mutated DNA was detected in all five KRAS positive specimens when cfDNA was extracted with the QIAamp circulating nucleic acid or Maxwell RSC ccfDNA Plasma Kit, it was detected in only the two specimens with the highest mutational load when cfDNA was extracted with the second version of the NEXTprep-Mag ccfDNA Isolation Kit and was not detected in any specimens extracted with the PME free-circulating DNA Extraction Kit, the first version of the NEXTprep-Mag ccfDNA Isolation Kit, or the EpiQuick Circulating Cell-Free DNA Isolation Kit. 

   Although both GAPDH fragments were amplified in all specimens when DNA was extracted with 5 of the 6 kits, when DNA was extracted using the EpiQuick Circulating Cell-Free DNA Isolation Kit only two specimens allowed for amplification and only one fragment was amplified in each.  The difference in amplification between the 105 bp and 236 bp fragment was similar for DNA isolated with the QIAamp circulating nucleic acid or Maxwell RSC ccfDNA Plasma Kit (-9.898 and -8.427, respectively). Compared to the QIAamp circulating nucleic acid, the differences between the 105 and 236 bp fragments were smaller when isolated with the EpiQuick Circulating Cell-Free DNA Isolation Kit (-4.894, P=0.001), the first and second versions of the NEXTprep-Mag ccfDNA Isolation Kit (-4.261, P<0.001 and -4.177, P<0.001, respectively), and the PME free-circulating DNA Extraction Kit (-4.491, P<0.001). 

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	EpiQuick Circulating Cell-Free DNA Isolation Kit First version of the NEXTprep-Mag ccfDNA Isolation Kit Second version of the NEXTprep-Mag ccfDNA Isolation Kit PME free-circulating DNA Extraction Kit QIAamp circulating nucleic acid kit Maxwell RSC cfDNA Plasma Kit
   Digital PCR Specific	Targeted nucleic acid	Total cfDNA KRAS mutated cfDNA
   Real-time qPCR Specific	Technology platform	Digital droplet PCR
   Real-time qPCR Specific	Targeted nucleic acid	GAPDH
   Real-time qPCR Specific	Length of gene fragment	105 bp 236 bp

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