Biotinylated DNA Probe Pulldown Assay

The pull-down assay was performed as described in previous study.9 (link) In brief, 10⁷ cells were washed with cold PBS, lysed in 500 μL co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μL washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction for 1 h at room temperature. Then, the RNAs were purified using RNeasy Mini Kit (QIAGEN) for further qPCR analysis.

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Huang W., Huang F, & Feng C. (2020). CircFoxo3 Promotes Adriamycin Resistance Through Regulation of miR-199a-5p/ATP Binding Cassette Subfamily C Member 1 Axis in Hepatocellular Carcinoma. OncoTargets and therapy, 13, 5113-5122.

Publication 2020

Streptavidin C1 magnetic beads RNeasy Mini Kit

Assay Buffer Cells Cold Dna probes Oligo Pull Rnas Streptavidin

Corresponding Organization : Central South University

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Variable analysis

independent variables

Biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3

dependent variables

Purified RNAs for further qPCR analysis

control variables

10^7 cells washed with cold PBS
Cells lysed in 500 μL co-IP buffer
Incubation with biotinylated DNA oligo probes at room temperature for 2 h
Addition of 50 μL washed Streptavidin C1 magnetic beads for 1 h at room temperature

controls

Positive control: Endogenous circ-Foxo3
Negative control: Not explicitly mentioned

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