Allprep dna rna nano kit

Eighteen milliliters of EDTA blood was collected and CTCs were isolated in duplicate from 5 ml of whole blood by positive immunomagnetic selection (AdnaTest EMT-2/StemCell Select^TM, QIAGEN) [13 (link)]. CTC-depleted blood remaining after positive immunomagnetic selection [20 (link)] as well as the remaining blood (not used for CTC isolation) were centrifuged and stored. EVs were isolated from pre-filtered plasma by affinity-based binding to a spin column [13 (link), 21 (link)]. Subsequently, the total RNA was isolated and purified (exoRNeasy Kit, QIAGEN). The mRNA was isolated from the CTC lysates and from the vesicular RNA eluates by Oligo(dT)₂₅ beads [13 (link)]. The supernatant remaining from the CTC lysates after incubation with the Oligo(dT)₂₅ beads, called the mRNA-depleted CTC lysate, was used to isolate the gDNA using the AllPrep DNA/RNA Nano Kit prototype (QIAGEN) [14 (link)]. cfDNA was isolated by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN) using plasma from CTC-depleted blood [20 (link)]. Buffy coat DNA and normal tissue DNA that was available (from 18 of the 26 patients) was used as matched germline control. Detailed protocols are available in Additional file 2.

The mRNA-depleted CTC lysates were pooled in cases where two lysates were available from the CTC isolation in duplicate (n = 16). The total volume of the mRNA-depleted CTC lysates was used to isolate the gDNA by a newly established workflow called the AllPrep DNA/RNA Nano Kit prototype (QIAGEN GmbH, Germany). Briefly, the lysates were mixed with 200 µL AdnaTest DNA Binding Buffer and 20 µL proteinase K and incubated for 10 min at 56 °C. After the addition of 200 µL ethanol, the whole mixture was applied to a QIAamp MinElute Spin Column. The membrane was washed using 500 µL AdnaTest DNA Wash Buffer A and 500 µL AdnaTest DNA Wash Buffer B. The membrane was then dried by centrifugation and the gDNA was eluted with 23 µL AdnaTest DNA Elution Buffer.