The biotin-labeled circBACH2 probe and control probe were obtained from RiboBio (China) commercially. The lysates of the MDA-MB-231 or BT-549 cells were obtained using Pierce™ IP Lysis Buffer (Thermo Fisher) and incubated with the 3 µg biotin-labeled circBACH2 probe or control probe at 4 °C overnight. The mixture was incubated with 50 µl streptavidin magnetic beads (Invitrogen, USA) for 3 h at 4 °C. Subsequently, the complex bound by the biotin-labeled circBACH2 probe or control probe was eluted, followed by qRT-PCR.
Control probe
Manufactured by RiboBio
Sourced in China
Control probes are laboratory reagents used to validate the performance of various analytical techniques. They serve as references to ensure the accuracy and reliability of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Vazyme (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Maxiscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Purebinding rna protein pull down kit, by Geneseed (1 mentions)
Streptavidin coated magnetic beads, by New England Biolabs (1 mentions)
Magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin coated magnetic beads, by Thermo Fisher Scientific (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using control probe
1
Biotin-labeled circBACH2 Probe Pulldown
2
Biotinylated miRNA Affinity Purification
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Cells were transfected with 80 nM biotinylated miR-23a/b mimics (miR-23a/b probes) or control probe (RiBoBio, Guangzhou, China) and were then harvested in lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.5% NP40 and 200 U/ml RNase inhibitor (Vazyme)). The lysate was next incubated with streptavidin magnetic beads (Invitrogen) at 4 °C overnight with constant rotation. After incubation, the beads were washed with lysis buffer four times, and RNA was extracted with TRIzol (Invitrogen) for analysis.
3
Characterization of circROBO1-miR-217 Interaction
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The biotinylated circROBO1 probe (5′-CGAAGTACTTGAACAGTCAG-3′) and control probe (5′-GAAACTGCTCGGAACGTTAA-3′) were synthesized by RiboBio (Guangzhou, China), while the pre-ROBO1 probes were transcribed using the MAXIscriptTM T7 Transcription Kit (Invitrogen, CA, USA). Then, an RNA-Protein Pull Down Kit (Thermo Fisher, MA, USA) was used according to the manufacturer’s guidelines. The abundance of hsa-miR-217-5p was detected by RT-qPCR, while the enriched protein was analyzed by western blot.
4
Linc-ROR and DEPDC1 3'UTR Interaction Assay
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RNA pull-down assays were performed using a PureBinding TM RNA-Protein Pull-down Kit (Guangzhou Geneseed Biotech Co., Ltd; cat. no. P0201) according to the manufacturer’s instructions. Biotin-labeled Linc-ROR, the DEPDC1 3′UTR probe (100 pmol) and the control probe (100 pmol) were synthesized by RiboBio (Guangzhou, China). Cells were lysed and incubated with 100 μL of streptavidin-coated magnetic beads for 30 min at 4 °C, with rotation at 10 rpm/min, with specific Linc-ROR or DEPDC1 probes. The cell lysates were incubated with streptavidin-coated magnetic beads to pull down the biotin-labeled RNA complex, or by RIP buffer with protease inhibitor cocktail. The abundance of Linc-ROR and miR-130a-3p was analyzed by qRT-PCR, or the obtained products were used for western blot detection mass spectrometry. The probe sequences are listed in Supplementary Table S3 .
5
RNA Pull-Down Assay for Enrichment
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RNA pull-down assay was done as previously described [25 (link)]. Biotin-labeled HCG11 probes and control probes were synthesized by RiboBio. Then, the probes were cultured with cell lysates for RNA enrichment. 30 μL of streptavidin magnetic beads (88816) was added into the samples for precipitation. At last, RNAs bound in the precipitated mixture were eluted and analyzed by qRT-PCR.
6
Enrichment of miR-526b-3p in U87 and U251 cells
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Probes, including biotinylated miR-526b-3p WT and biotinylated miR-526b-3p Mut, or control probes purchased from RiboBio (Guangzhou, China) were transfected into U87 and U251 cells. Then, DNaseI was added to RNA solution, and incubation was for 5 min at 65°C. Afterwards, streptavidin-coated magnetic beads (New England BioLabs, USA) were incubated for another 4 h. Finally, the beads were washed with PBS and extracted RNA. MiR-526b-3p enrichment was detected via RT-qPCR analysis [19 (link)].
7
Biotin-labeled miRNA Pulldown Assay
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The biotin-labelled miRNA pulldown assay was performed using a Magnetic RNA-protein pulldown kit (Thermo Fisher Scientific), with some modifications to the manufacturer’s instructions. The biotin-labelled miR-125a-5p (5′-UCCCUGAGACCCUUUAACCUGUGA-3′) and the control probes (5′-AACGCTTCACGAATTTGCGT-3′) were purchased from RiboBio Company (Guangzhou, China). Briefly, biotin-labelled miR-125a-5p or the control probes were transfected into LUAD cells at a final concentration of 30 nM. After incubation for 48 h, the cells were lysed using RIPA lysis buffer containing RNase inhibitors and centrifuged at 10,000
g for 10 min. To capture the mRNA bound to miR-125a-5p, the supernatant was incubated with streptavidin beads for 30 min with agitation at room temperature. Then, the unbound RNAs were washed off with 100 μL of wash buffer three times. Total RNA was isolated using 50 μL elution buffer, and the RNA eluates were analyzed by quantitative RT-PCR.
g for 10 min. To capture the mRNA bound to miR-125a-5p, the supernatant was incubated with streptavidin beads for 30 min with agitation at room temperature. Then, the unbound RNAs were washed off with 100 μL of wash buffer three times. Total RNA was isolated using 50 μL elution buffer, and the RNA eluates were analyzed by quantitative RT-PCR.
8
Affinity Purification of miR-223-3p Targets
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Probes, including biotinylated miR-223-3p mimic, biotinylated miR-223-3p Mut, and control probes purchased from RiboBio (Guangzhou, China) purchased from RiboBio (Guangzhou, China) were transfected into A549 and SAECs cells. Then DNaseI was added to RNA solution, and incubation was for 5 min at 65 °C. Afterwards, streptavidin-coated magnetic beads (Invitrogen, Carlsbad, CA, USA) were incubated for 4 h. qRT-qPCR was carried out to analyze the enrichment of the co-deposited PARP-1 RNA.
9
Biotin-labeled RNA Pulldown Assay
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CircStag1 probes with biotin labels and control probes were synthesized by RiboBio Biotechnology (Table S5 ). Biotin-labeled RNA pulldown was performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo, USA) following the manufacturer’s instructions. Briefly, the probes (50 pmol·L−1) were incubated with streptavidin-coated magnetic beads for 30 min at room temperature. The cells were lysed with Thermo Scientific Pierce IP Lysis Buffer (Thermo, USA), incubated with the probe-bound magnetic beads and rotated for 1 h at 4 °C. Then, the probe-bead-protein complex was washed three times with washing buffer. The proteins in the complex were extracted and detected by western blotting.
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