QIAsymphony DSP Circulating DNA Kit

For automated purification of ccfDNA from 1-96 samples using the QIAsymphony SP

Features

For in vitro diagnostic use
Lot-to-lot traceability
Produced under GMP manufacturing conditions

Product Details

The QIAsymphony DSP Circulating DNA Kit provides reagent cartridges and reagents for fully automated and simultaneous purification of human circulating cell-free (ccf) DNA from human plasma and urine using the QIAsymphony SP instrument. Protocols are available for 2 and 4 ml sample volumes.

Principle

QIAsymphony technology combines the speed and efficiency of anion exchange-based nucleic acid purification with the convenient handling of magnetic particles (see figure “ Schematic diagram of the QIAsymphony SP principle.”).

The QIAsymphony SP processes a sample containing magnetic particles as follows: 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. These steps are repeated several times during sample processing The QIAsymphony SP uses a magnetic head containing an array of 24 magnetic rods, and can therefore process up to 24 samples simultaneously.

See figures

Efficient processing of magnetic particles.

Procedure

The QIAsymphony SP makes automated sample preparation easy and convenient. Samples, reagents and consumables, and eluates are separated in different drawers. Simply load samples, proteinase K, reagents provided in special cartridges, and preracked consumables in the appropriate drawer before a run. Start the protocol and remove purified DNA from the “Eluate” drawer after processing. Refer to the user manuals supplied with your instrument for operating instructions.

The purification procedure is designed to ensure safe and reproducible handling of potentially infectious samples, and comprises 3 steps: bind, wash, and elute (see figure “ QIAsymphony DSP Circulating DNA procedure.”). The user can choose between different sample input volumes.

See figures

QIAsymphony DSP Circulating DNA procedure

Applications

ccfDNA purified using the QIAsymphony DSP Circulating DNA Kit and the QIAsymphony SP is ready for use in a wide range of downstream applications, including NGS, digital, multiplex and quantitative real-time PCR.

Supporting data and figures

Efficient processing of magnetic particles.

The QIAsymphony SP processes a sample containing magnetic particles as follows. A magnetic rod protected by a rod cover enters a well containing the sample and attracts the magnetic particles. The magnetic rod with cover is positioned above another well and releases the magnetic particles. The QIAsymphony SP uses a magnetic head containing an array of 24 magnetic rods, and can therefore process 24 samples simultaneously. Steps 1 and 2 are repeated several times during sample processing.

Resources

FAQ

Human plasma that has been collected in EDTA or citrate 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.

FAQ ID - 3702

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699

Open reagent cartridges can be used for up to 4 weeks without affecting performance. After 4 weeks, circulating cell-free DNA yields are expected to decrease. To ensure the best performance, carefully seal buffer troughs of a partially used reagent cartridge with Reuse Seal Strips when not in use; incomplete sealing of reagent cartridge can reduce the performance.

FAQ ID - 3706

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.

FAQ ID - 3701

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).

FAQ ID - 3700

Generation of size-distribution profiles for circulating cell-free DNA (ccfDNA) from eluates can be used to check the quality of fragmented ccfDNA and potential genomic DNA background. However, due to the limited sensitivity of these assays, they are not recommended for quantification of ccfDNA. Samples with low ccfDNA eluate concentration and, thus, low ccfDNA yields might exhibit no peak at approximately 165 bp in size-distribution profile results.

FAQ ID - 3705

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.

FAQ ID - 3704

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.

FAQ ID - 3703