QIAsymphony DSP Circulating DNA Kits

Sample volumes of 2 ml and 4 ml can be used without affecting the number of preps per kit (i.e., 192 samples per kit).

At least 2.4 ml sample for the 2 ml protocol or 4.5 ml for the 4 ml protocol should be loaded.

Loading smaller volumes increases the risk of “unclear” flagged samples. As described in the corresponding labware list, volumes of 1.4–2.4 ml and 3.5–4.5 ml, respectively, will be processed but flagged “unclear” (“Enable less sample” mode).

Loading volumes less than recommended increases the risk that samples are not transferred (“invalid” flagging). If the sample volume is insufficient, add PBS to the required sample volume before loading the sample.

 When using FIX labware (instrument does not perform liquid-level detection and reporting), load at least 2.1 ml for the 2 ml protocol and at least 4.1 ml for the 4 ml protocol. Because the instrument does not detect sample volumes using FIX labware,  insufficient volumes might result in bubble formation during sample transfer and/or the subsequent binding step. If the sample volume is insufficient, add PBS to the required sample volume before loading the sample.

Human plasma that has been collected in EDTA blood collection tubes or ccfDNA stabilized blood collection tubes can be used as sample material. In addition, human urine (non-stabilized or stabilized) can also be used.

Serum is not recommended as sample material because it contains a large background of genomic DNA.

When an elution volume of 60 µl is selected, the instrument transfers 75 µl elution buffer to ensure that at least 60 µl eluate is available for downstream analysis. In most cases, the eluate volume is 60–70 µl, depending on temperature, humidity and how long the eluate remains on the instrument before being removed. To calculate total yield or recovery of an internal control, an elution volume of 75 µl should be used.

Typically, a yield of 7.5 ng/ml plasma or urine can be expected. This yield results in <1 ng/µl eluate. However, the yield will vary from sample to sample, due to large donor-dependent variations in circulating cell-free DNA (ccfDNA) concentration in plasma: variation can be approximately 20-fold for healthy blood donors and 1000-fold for both clinical samples and urine.

Because of the very low concentrations of ccfDNA in sample materials (e.g., 0.1 ng/µl eluate is not unusual), measurement of DNA with a spectrophotometer is not recommended. A sensitive and accurate fluorescence-based quantitation assay or a real-time PCR assay should be used for ccfDNA-concentration determination.