Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of methods for isolation and quantification of circulating cell-free DNA from patients with endometriosis.

Author(s): Huebner H, Lubrich H, Blum S, Antoniadis S, Lermann J, Ekici A, Fasching PA, Beckmann MW, Ruebner M, Burghaus S

Publication: Reprod Biomed Online, 2021, Vol. , Page

PubMed ID: 34493460 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared ccfDNA yield and integrity and mitochondrial ccfDNA yield after ccfDNA isolation from plasma using either the Maxwell kit or the QIAamp kit. The ccfDNA yield was measured using fluorometry as well as real-time PCR amplification of ALU. Blood was collected into Streck cell-free DNA blood collection tubes from thirty-two women (ages 18-48) with endometriosis who were undergoing laparoscopy.  After overnight storage at room temperature, plasma was obtained by centrifugation at 1600 g for 10 min followed by 14000 g for 10 min. ccfDNA was extracted from plasma using the Maxwell RSC ccfDNA plasma kit or the QiAamp minElute ccfDNA mini kit and stored at -20°C. ccfDNA was quantified with the QuantiFluor RNA System kit. ccfDNA fragment size was evaluated by real-time PCR amplification of 60 bp and 247 bp fragments of ALU. Mitochondrial ccfDNA was quantified by amplification of a 64 bp fragment of mitochondrial DNA.  

   Summary of Findings:

   The mean concentration of ccfDNA per mL of plasma was higher when extraction was with the Maxwell rather than QIAamp kit (7.37 ng/mL versus 4.45 ng/mL, P<0.0001), with higher yields obtained with Maxwell kit from 31 of the 34 plasma specimens. The DNA yields as measured by fluorometry were modestly correlated between the two extraction methods (r=0.6995; P<0.0001). There was also a significantly higher number of copies per mL of plasma of both the 60 bp and 247 bp fragments of ALU when extraction was with Maxwell kit compared to the QIAamp kit (P<0.0001 and P<0.0060, respectively), but the number of copies of each amplicon per ng of cfDNA were comparable between extraction methods. Fluorometric and real-time PCR quantified levels of the 60 bp amplicon of ALU were modestly to strongly correlated regardless of extraction kit (r=0.677 for QIAamp and r=0.8158 for Maxwell; P<0.0001, both). The DNA integrity, measured by the ratio of 247 bp to 60 bp fragments of ALU, was superior among specimens extracted using the QIAamp kit in comparison to the Maxwell kit (0.6444 versus 0.5197, P<0.0001); in fact, 28 of the 34 plasma specimens had a higher ratio of 247 bp to 60 bp ALU fragments following extraction with the QIAamp kit compared to the Maxwell kit. The number of copies of mitochondrial cfDNA per mL of plasma was higher when extraction was with the Maxwell kit than the QIAamp kit (P=0.018), but the difference in copy number per ng of ccfDNA was not significant.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Streck/BCT

   Diagnoses:

   • Endometriosis

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Maxwell RSC ccfDNA plasma kit QiAamp minElute ccfDNA mini kit
   Real-time qPCR Specific	Technology platform	Real-time PCR QuantiFluor
   Real-time qPCR Specific	Targeted nucleic acid	ALU Mitochondrial ccfDNA
   Real-time qPCR Specific	Length of gene fragment	60 bp 247 bp

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