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Liquid biopsy

Circulating cell-free DNA

Tissue biopsies remain the standard for diagnosis but they only represent one point in time and are limited by repeatability, cost and time. ccfDNA overcomes this by providing real-time mutational information that can be used to detect and monitor biomarkers of cancer and other diseases in a simple blood test.
Since ccfDNA is often found in low concentrations with rare mutations or in a fragmented condition, you need reliable tools that give you confidence in the signal you detect. Whether you work in biomarker discovery, transplant or NIPT research – we offer gold-standard solutions, for ccfDNA sample preparation and analysis, with the highest quality assurance.

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General

 

What is ccfDNA?

Many studies have shown that elevated levels of ccfDNA also known as ctDNA, can be found in the blood of patients suffering from various diseases, especially cancer. ccfDNA differs from normal cell-free DNA by the presence of somatic mutations, which can be used as genetic markers to determine therapeutic response and guide treatment decisions. Levels of mutant ccfDNA can also be useful for following-up cancer patients and detecting minimal residual disease.

 

How can I overcome the challenges of isolating ccfDNA?

Our QIAamp MinElute ccfDNA and EZ1 ccfDNA Kits use a unique bead and column based method to reliably deliver high-quality material from human plasma or serum for research by providing: efficient recovery of fragmented DNA, scalable sample input volume of 1–10 ml, no organic extraction or ethanol precipitation, effective removal of contaminants and inhibitors.

 

Can I isolate more than one analyte from one sample?

The QIAamp ccfDNA/RNA Kit offers co-isolation of ccfDNA and ccfRNA from the same plasma sample, providing excellent yield and quality of both analytes in the same eluate.
This allows you to analyze both DNA and RNA mutations in the same liquid biopsy sample or can boost detection sensitivity to discover low-frequency tumor mutations for research.

Sample collection

 

What is the minimum volume of blood to draw?

With most kits on the market, 8–10 ml of blood is required from each draw to further process the plasma and ccfDNA. Similarly with the PAXgene Blood ccfDNA Tubes, a blood draw of 10 ml is required. This measure is to ensure a 0.15 ml ratio of cell stabilization additive per ml of blood when the evacuated tube is filled correctly.

 

When you talk about blood collection, does that mean full blood, serum or plasma?

For sample collection, the PAXgene Blood ccfDNA Tubes are for the collection of whole human blood. Subsequently, you can use a protocol with the tubes to process plasma. ccfDNA can then be purified manually, using the QIAamp Circulating Nucleic Acid Kit – or automatically, using the PAXgene Blood ccfDNA Kit.

 

Can you use butterfly needles for blood draw with the PAXgene Blood ccfDNA tubes?

Yes, the PAXgene Blood ccfDNA Tubes can be used with standard blood collection equipment. You can also refer to the PAXgene Blood ccfDNA Tube handbook for more details on sample collection procedures.

Sample stabilization and preparation

 

What kind of blood stabilization should be used for DNA extraction and analysis via PCR?

It is preferable if you can prevent DNA modification from crosslinking reagents. The PAXgene Blood ccfDNA Tubes use an alternative, proprietary solution for stabilization that does not include crosslinking. This solution lets you decrease the release of gDNA and minimize hemolysis – effectively preserving the integrity of your sample – while also preventing DNA modification, which can complicate PCR results.

 

Are DNA methylation states maintained in blood?

Yes, ccfDNA purified with the PAXgene Blood ccfDNA System is compatible with methylation-specific assays that require treatment with bisulfite. In this poster, we also compared the performance of EDTA, Streck and PAXgene Blood ccfDNA Tubes at two time points – 0 days and 6 days – using the Agilent Bioanalyzer. We saw after 6 days that the Streck samples had a 7–8 bp shift in the main ccfDNA peak, which indicates DNA modification by formaldehyde-releasing reagents.

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