Experiment

To investigate the effect of room temperature (22⁰C) storage of blood samples on tumor DNA concentrations over a 28 day period in blood drawn into Blood Exo DNA ProTeck® compared to a K3EDTA tube.

Conclusion

Blood Exo DNA ProTeck® tubes stabilize tumor DNA in blood for up to 28 days at room temperature (22⁰C).

Methods
  • Blood drawn

    Blood was drawn into a K3EDTA (10 mL) and Blood Exo DNA ProTeck® (10 mL) tubes from healthy donors.

  • Spike cell-free DNA

    A known amount of KRAS exon 2 G13D tumor cell-free DNA obtained from a tumor cell line was spiked into all blood samples and toughly mixed.

  • Time series analysis

    After mixing blood was aliquoted into five equal aliquots and stored at 22°C. At indicated time points plasma cell-free DNA was extracted from aliquots for analysis.

  • Detect KRAS DNA

    Wildtype KRAS DNA was detected using Bio-Rad PrimePCR ddPCR mutation assay: KRAS WT assay (cat. # 178441544).

  • Extract cell-free DNA

    Cell-free DNA extracted from sample aliquots were analyzed for KRAS exon 2 G13D mutation using therasreen KRAS RGQ PCR kit and Rotor Gene real time PCR instrument.

  • Quantify cell-free DNA concentrations

    Quantification of KRAS wildtype DNA concentration using Bio-Rad PrimePCR ddPCR mutation assay: KRAS WT assay.

ProTeck tube vs K3EDTA tube
cancer stability
Observation

Our data show that wildtype KRAS DNA concentration increases in K3EDTA blood over time whereas in ProTeck tube wildtype KRAS concentration was stable at 22°C for up to 28 days.

ProTeck tube vs K3EDTA tube
K3EDTA Day 0 Day 3 Day 7 Day 14 Day 28
Donor 1 Positive Negative Negative Negative Negative
Donor 2 Positive Negative Negative Negative Negative
Donor 3 Positive Negative Negative Negative Negative

ProTeck Day 0 Day 3 Day 7 Day 14 Day 28
Donor 1 Positive Positive Positive Positive Positive
Donor 2 Positive Positive Positive Positive Positive
Donor 3 Positive Positive Positive Positive Positive
Observation
  1. KRAS exon 2 G13D mutated DNA could be detected in blood drawn into ProTeck tubes even after 28 days at 22°C (room temperature).

  2. Data show that KRAS exon 2 G13D mutated DNA in K3EDTA blood could be detected only at day 0.

  3. Due to high wildtype background DNA, mutated DNA could not be detected in K3EDTA blood after day 0.

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