eBook: Optimizing Results from Nucleic Acid Isolation - 6

standardization, which are absolutely essential if
adoption of liquid biopsies is to enter mainstream
medical diagnostics.

group cannot be compared with those conducted
at another location. Variability in sample processing
protocols, extraction methods, and approaches to
quantification still introduce significant lack of comparability across results. Recently, progress has been
made to better define factors influencing preanalytical sample processing and storage, but variability
between extraction methods and quantification approaches still introduces significant sources of error.

Assuming best practices in blood collection, fractionation, and storage, yield and quality bottlenecks
shift over to the extraction step, where it can be said
that the product is the process. Yield, quality, cellular genomic contamination levels, and fragment
length are highly dependent on using the right collection tubes, anticoagulants, processing times and
temperatures, tube agitation, centrifugation protocols, intermittent storage conditions, and cfDNA extraction and quantification methods, which in turn
affect downstream analysis.

Cytiva has engineered its cfDNA purification kits to
address these concerns. Its Sera-Xtracta™ Cell-Free
DNA Kit, for example, uses chaotropic agents to
disrupt binding between target nucleic acids and
contaminants. Sera-Xtracta provides efficient extraction and purification of cfDNA from very small
plasma samples as the front-end for liquid biopsies
and other studies. The kit enables capture of smaller cfDNA fragments for increased sensitivity, while
minimizing co-purification of higher-molecular
weight genomic DNA. Moreover, the kits are compatible with modern molecular methods, including
NGS, qPCR, ddPCR, BEAMing, and other amplification and genotyping applications.

Extraction should be designed to purify all cfDNA
species to the same degree and clear all contaminants equally, as isolation and extraction methods
affect assay sensitivity for both cfDNA yield and
quality. Sample prep should proceed as rapidly as
possible in standard collection tubes, and for up to
six hours in tubes containing preservatives. Care
should be taken to minimize the release of genomic
DNA from ruptured cells. Samples intended for longterm storage should be kept at −80°C, but even then
some degradation should be expected.

About the author
Angelo DePalma earned his Ph.D. in organic chemistry
from Stony Brook University and was previously senior
scientist at Schering-Plough. He has written extensively
on biotechnology, biomanufacturing, medical devices,
pharmaceutical commerce, laboratory instrumentation, and advanced materials.

cfDNA analysis has become possible, and even routine, thanks to advances in molecular detection techniques such as PCR and derivative methods, NGS,
and genome-wide sequencing. But without method inter-assay compatibility, results from one lab or

https://www.ncbi.nlm.nih.gov/pubmed/32083442 https://www.ncbi.nlm.nih.gov/pubmed/32083442 https://www.ncbi.nlm.nih.gov/pubmed/21861994 https://www.ncbi.nlm.nih.gov/pubmed/21861994 https://www.ncbi.nlm.nih.gov/pubmed/21861994 https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=32777 https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=32777 https://www.cytivalifesciences.com/shop/molecular-biology/purification/magnetic-beads/sera-xtracta-cell-free-dna-kit-p-11561 https://www.ncbi.nlm.nih.gov/pubmed/32122922 https://www.gene-quantification.de/Johansson-Stahlberg-et-al-BDQ-SI-2019.pdf https://www.nature.com/articles/s41598-018-23766-9 https://www.nature.com/articles/s41598-018-23766-9

eBook: Optimizing Results from Nucleic Acid Isolation

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