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QIAsymphony PAXgene Blood ccfDNA Kit

For purification of ccfDNA from plasma using the QIAsymphony SP

  • Optimized chemistry and input volumes for PAXgene Blood ccfDNA Tubes
  • Separate protocol lines for small (STA) and large (LAF) ccfDNA fragments
  • Prefilled reagent cartrdiges with bar code for safety and ease of use
  • Standardized processing for high reproducibility

 


The PAXgene Blood ccfDNA System consists of a blood collection tube (PAXgene Blood ccfDNA Tube) and a ccfDNA purification kit for the QIAsymphony (QIAsymphony PAXgene Blood ccfDNA Kit).
Cat No./ID: 768536
QIAsymphony PAXgene Blood ccfDNA Kit (192)

Reagent cartridges, accessories and proteinase K vials for 192 preps

For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
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Isolated ccfDNA is compatible with methylation-specific downstream applications.

Whole blood of 4 healthy donors was collected into either EDTA or PAXgene Blood ccfDNA Tubes. Blood was left at room temperature for up to 7 days. Each day, ccfDNA was isolated from samples, bisulphite converted (Epitect FAST kit, QIAGEN) and analyzed with the GEMINI assay (Clinical Genomics) based on 2 methylated (BCAT1 and IKZF1) and 1 control (ACTB) target. Three PCR replicates per target sequence were analyzed. ccfDNA from blood stored in EDTA tubes generated more false positives than from blood stored in PAXgene Blood ccfDNA Tubes (data courtesy of Clinical Genomics).

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Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs.

With the standard protocols (STA), predominantly small ccfDNA fragments are isolated. The large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both protocol lines consist of one protocol for 2.4 ml and one protocol for 4.8 plasma input.

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PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time.

The change in delta-CT values of target versus control signal for ccfDNA isolated from whole blood stored in EDTA and PAXgene Blood ccfDNA Tubes was measured at different storage intervals. Shown are the relative signals of the 3 mutations covered by the therascreen EGFR assay: [A] point mutation T790M, [B] deletion in exon 19 and [C] L858R. Each PCR was done in duplicate. To calculate the delta-CT value, the average CT of each mutant was subtracted from the average CT of the wildtype sub-assay. The average of all donors is shown in the figure for all tested time points. The therascreen EGFR assay defines a threshold for each sub-assays. Delta-CT values above this threshold result in false-negative calls. Results from the ccfDNA isolated from EDTA tubes exceeded this threshold over time whereas results from the PAXgene Blood ccfDNA Tubes remained stable.

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The QIAsymphony SP processes samples containing magnetic particles.

A magnetic rod protected by a rod cover enters a well containing the sample and attracts the magnetic particles. The magnetic rod cover is positioned above another well and the magnetic particles are released. The QIAsymphony SP uses a magnetic head with an array of 24 magnetic rods, and can therefore process up to 24 samples simultaneously. Collection, transfer and release of magnetic particles is repeated several times during sample processing.

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High performance, comparable to QIAamp Circulating Nucleic Acid Kit.

Results from two studies assessing the performance of the QIAsymphony PAXgene Blood ccfDNA Kit. Study A compares ccfDNA yield and portion of double-stranded DNA of eluates generated with the QIAsymphony PAXgene Blood ccfDNA Kit to eluates from the manual QIAamp Circulating Nucleic Acid Kit. Study B summarizes performance parameters for the PAXgene ccfDNA system. The QIAsymphony PAXgene Blood ccfDNA Kit provides consistently accurate results.

Performance

The fully automated QIAsymphony PAXgene Blood ccfDNA Kit procedure generates ccfDNA yields comparable to the manual QIAamp Circulating Nucleic Acid Kit procedure (see figure High performance, comparable to QIAamp Circulating Nucleic Acid Kit). Since yield is strongly dependent of donor, individual yields may vary. Onboard measures of the QIAsymphony SP minimize cross-contamination.

Principle
Apoptosis and lysis of white blood cells in unstabilized whole blood releases genomic DNA into plasma, which dilutes naturally occurring ccfDNA. PAXgene Blood ccfDNA Tubes contain an additive that prevents blood coagulation and immediately stabilizes blood cells via a non-crosslinking stabilization solution (see figure PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time). This impedes the release of intracellular DNA and maintains constant ccfDNA levels in plasma. When used with PAXgene Blood ccfDNA Tubes, the proven magnetic particle technology of the QIAsymphony PAXgene Blood ccfDNA Kit provides a rapid, automated procedure to isolate ccfDNA of high quality and purity (see figure Isolated ccfDNA is compatible with methylation-specific downstream applications).
Procedure

The workflow begins with a centrifugation step that separates plasma from the cellular fraction of the whole blood collected into PAXgene Blood ccfDNA Tubes. The plasma is then carefully transferred into a second tube. A second centrifugation step can be carried out optionally to remove any traces of buffy coat. The pure plasma is then transferred to a third tube which is loaded into the QIAsymphony SP.

In the QIAsymphony SP, plasma proteins are digested by proteinase K while ccfDNA binds to the surface of magnetic beads (see figure The QIAsymphony SP processes samples containing magnetic particles). Depending on the chosen protocol line, predominantly small ccfDNA fragments (STA protocol line) or small and large ccfDNA fragments (LAF protocol line) are isolated. Three wash steps ensure contaminant removal. Finally, ccfDNA is eluted from the magnetic particles and is ready for use in downstream applications (see figure Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs).

Applications

ccfDNA purified using the QIAsymphony PAXgene Blood ccfDNA Kit and the QIAsymphony SP is ready for use in a wide range of downstream applications, including:

  • PCR, digital, multiplex and quantitative real-time PCR 
  • Pharmacogenomic studies 
  • SNP genotyping
  • Next generation sequencing
  • Methylation assays

 

Features
Specifications
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Application/Protocol Documents (1)
For purification of ccfDNA from human whole blood stabilized in PAXgene Blood ccfDNA Tubes using the QIAsymphony SP and QIAsymphony PAXgene Blood ccfDNA Kit.

 


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Kit Handbooks (1)
For purification of ccfDNA from plasma using the QIAsymphony SP
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