Isolation and Characterization of Circulating Tumor Cells

Blood was collected and processed as previously described [35 (link), 51 (link)]. Briefly, whole blood collected by venipuncture into EDTA tubes was separated by centrifugation with Ficoll-Paque PLUS (Fisher Scientific). The layer containing mononucleated cells was depleted of CD45+ cells by magnetic LS MACS columns (Miltenyi). CTCs were isolated using an anti-EpCAM goat polyclonal antibody (R&D Systems). RNA was isolated using oligo (dT) Dynabeads (Invitrogen), and DNA was isolated using MagneSil Paramagnetic Particles (PMPs) (Promega) as previously described [34 (link)].

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Rodems T.S., Juang D.S., Stahlfeld C.N., Gilsdorf C.S., Krueger T.E., Heninger E., Zhao S.G., Sperger J.M., Beebe D.J., Haffner M.C, & Lang J.M. (2022). SEEMLIS: a flexible semi-automated method for enrichment of methylated DNA from low-input samples. Clinical Epigenetics, 14, 37.

Publication 2022

Ficoll-Paque PLUS magnetic LS MACS columns oligo (dT) Dynabeads MagneSil Paramagnetic Particles (PMPs)

Anti antibody Blood Cells Centrifugation Edta Epcam Ficoll Goat Oligo Promega Venipuncture

Corresponding Organization :

Other organizations : University of Wisconsin Carbone Cancer Center, University of Washington, University of Wisconsin–Madison, Fred Hutch Cancer Center

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Variable analysis

independent variables

Anti-EpCAM goat polyclonal antibody (R&D Systems) used for CTC isolation

dependent variables

Isolated CTCs
Isolated RNA
Isolated DNA

control variables

Whole blood collected by venipuncture into EDTA tubes
Separation of blood by centrifugation with Ficoll-Paque PLUS (Fisher Scientific)
Depletion of CD45+ cells by magnetic LS MACS columns (Miltenyi)
RNA isolation using oligo (dT) Dynabeads (Invitrogen)
DNA isolation using MagneSil Paramagnetic Particles (PMPs) (Promega)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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