cfDNA purification application note

cfDNA isolation

Automated cfDNA isolation from plasma or serum

Cell-free DNA (cfDNA) extracted from plasma serum is gaining more and more attention as a biofluid sample type that fits vast application needs in the space of oncology research. Between the many applications relying on cfDNA analysis are the identification of actionable mutations and monitoring response to treatment; clinical research for non-invasive prenatal testing and diagnosis, known as NIPT and NIPD; and organ transplant monitoring research.

There are, however, numerous challenges related to cfDNA isolation, namely the difficulty in recovering cfDNA fragments due to their small size (less than 200 bp), the dilution of cfDNA with genomic DNA released from contaminating leukocytes, and the inherent sample variability for yield that tends to be very low. At PerkinElmer, we understand the importance of having a robust and reliable extraction process for cfDNA, since obtaining plasma and serum can potentially be limiting and difficult for a lab.

To give the lab the confidence of obtaining quality cfDNA they need from every extraction, PerkinElmer offers the NextPrep-Mag™ cfDNA automated isolation kits as an automated, high-throughput solution. These reagents have been validated for use on the chemagic™ 360 and Prime™ platforms to allow for high input of starting material and optimal extraction to help labs get quality cfDNA as a starting point for their downstream molecular applications.

Fig. 1: 
Analysis of cfDNA extracted with NextPrep-Mag™ cfDNA isolation kits on the chemagic™ 360 instrument.

cfDNA from EDTA plasma was extracted using the NextPrep-Mag™ cfDNA isolation kit with different inputs and compared to the NextPrep-Mag™ cfDNA automated isolation kit for yield. Results show that yield of cfDNA is linear to sample input.

Features of PerkinElmer cfDNA purification workflows

PerkinElmer cfDNA purification workflows, consisting in the combination of the NextPrep-Mag™ cfDNA chemistry and chemagic™ instruments, provide reliable isolation of high yields of cfDNA from up to 5 ml of plasma per sample. With only 15 minutes of hands-on time, PerkinElmer cfDNA workflows have a capacity of 24 samples in less than 90 minutes. The purified DNA is compatible with many downstream applications, such as producing cfDNA libraries with the NEXTFLEX® Cell-Free DNA-Seq kit as well as commercial amplicon panels.

Fig. 2: 
Sequencing results using cfDNA purified with a PerkinElmer workflow.

cfDNA extracted using the NextPrep-Mag™ cfDNA isolation kit was made into libraries for sequencing using the NEXTFLEX® Cell Free DNA-Seq kit. Reads were aligned to human genome GRCh38 and binned to insert size to examine the effect of size selection on mononucleosome and non-mononucleosome quantity. Y chromosome inserts represent reads derived from fetal DNA. Binned inserts show that size selection excludes non-mononucleosomal reads from sequencing data.

For more information

For more information, please download the application note and the poster on automated cfDNA purification.

For research use only. Not for use in diagnostic procedures.