LINC00526 full‐length sequences were in vitro transcribed and biotin‐labelled from pSPT19‐LINC00526 with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche) following their protocols. LINC00526 antisense RNA was in vitro transcribed and biotin‐labelled from pSPT19‐LINC00526 with the Biotin RNA Labeling Mix (Roche) and Sp6 RNA polymerase (Roche) following their protocols. After treatment with DNase I (Takara), the in vitro transcribed RNA was purified by the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Next, 3 µg of purified biotinylated RNA was incubated with 1 mg of U87 or U251 whole‐cell lysates at 25°C for 1 hour. Then, the complexes were isolated by streptavidin agarose beads (Invitrogen). The enriched proteins in the complexes were measured by Western blot.
Biotin rna labeling mix
Manufactured by Roche
Sourced in United States, Switzerland, Germany, China
Biotin RNA Labeling Mix is a reagent used for the incorporation of biotin labels into RNA molecules during in vitro transcription or reverse transcription reactions. It enables the detection and analysis of labeled RNA samples through various downstream applications, such as northern blotting, microarray hybridization, or pull-down experiments.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (171 mentions)
T7 rna polymerase, by Roche (138 mentions)
Streptavidin agarose bead, by Thermo Fisher Scientific (119 mentions)
Rnase free dnase 1, by Roche (72 mentions)
T7 rna polymerase, by Promega (26 mentions)
Rnase free dnase 1, by Promega (26 mentions)
Streptavidin beads, by Thermo Fisher Scientific (24 mentions)
T7 sp6 rna polymerase, by Roche (21 mentions)
Rna structure buffer, by Thermo Fisher Scientific (20 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (19 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (14 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (14 mentions)
T7 rna polymerase, by Takara Bio (13 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (12 mentions)
Rnase free dnase 1, by Takara Bio (11 mentions)
Sp6 rna polymerase, by Roche (11 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (11 mentions)
Rna immunoprecipitation wash buffer, by Thermo Fisher Scientific (10 mentions)
Rneasy plus mini kit, by Qiagen (9 mentions)
T7 polymerase, by Promega (9 mentions)
472 protocols using biotin rna labeling mix
1
LINC00526 Interactome Profiling in Glioma
2
lncSHRG-SATB1 Interaction Mapping by RNA Pulldown
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For RNA pulldown assays, Biotin-labeled lncSHRG and anti-sense control of lncSHRG were obtained by T7 transcription in vitro using Biotin RNA labeling Mix (Roche) and added into cell lysates containing His-SATB1. After incubation overnight at 4°C, Streptavidin-magnetic C1 beads was added and incubated for another 4 h at 4°C. Then biotin-enriched components were separated with SDS–PAGE and examined by WB. For lncSHRG domain mapping, truncated lncSHRGs were cloned into pCDNA3 plasmid. Then truncated lncSHRG DNA fragments were amplified by PCR and acted as templates for T7 transcription in vitro with Biotin RNA labeling Mix (Roche). The pulldown assays were perform as described above. For mass spectrometry, the biotin-enriched components were separated with SDS–PAGE and subjected to silver staining. Differential bands enriched by lncSHRG were collected for mass spectrometry (LTQ Orbitrap XL).
3
Identifying circOAS3-Interacting Proteins
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The interaction between circOAS3 and RNA-binding protein was detected by Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scienti c, USA) according to the manufacturer's protocols. Biotin-labeled probes targeting the junction site of circOAS3 were synthesized by RiboBio (Guangzhou, China) and a control probe was used as a control. Linear circOAS3 was transcribed with Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Thermo Fisher Scienti c, USA), circularized using T4 RNA ligase I and digested by RNase R. The cells were lysed and incubated with a biotin-labeled circOAS3 probe. Afterward, cell lysates were subjected to streptavidin agarose magnetic beads at normal temperatures. Finally, interacting proteins were identi ed by mass spectrometry and Western blot. The sequences of the probe were shown in Supplemental Table 2.
4
Identifying circOAS3-Interacting Proteins
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The interaction between circOAS3 and RNA-binding protein was detected by Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scienti c, USA) according to the manufacturer's protocols. Biotin-labeled probes targeting the junction site of circOAS3 were synthesized by RiboBio (Guangzhou, China) and a control probe was used as a control. Linear circOAS3 was transcribed with Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Thermo Fisher Scienti c, USA), circularized using T4 RNA ligase I and digested by RNase R. The cells were lysed and incubated with a biotin-labeled circOAS3 probe. Afterward, cell lysates were subjected to streptavidin agarose magnetic beads at normal temperatures. Finally, interacting proteins were identi ed by mass spectrometry and Western blot. The sequences of the probe were shown in Supplemental Table 2.
5
Protein-RNA Interaction Assay
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The interaction between KHDRBS3 and cDENND4C was tested by Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer’s instruction. In brief, Biotin-labeled cDENND4C (bio-cDENND4C) were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega), treated with RNase-free DNase I (Promega) and purified with RNeasy Mini Kit (QIAGEN). The bio-cDENND4C and bio-antisense RNA (NC) were incubated with GECs lysates followed by extensive washes. Magnetic beads were added to prepare a probe–magnetic bead complex. The retrieved proteins were analyzed by Western blot with GAPDH as the control.
6
LINC00662 Interactome Identification
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Wild‐type and miR‐15a/16/107 binding sites mutated LINC00662 were in vitro‐transcribed from pSPT19‐LINC00662 and pSPT19‐LINC‐mut, respectively, and biotin‐labeled using the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche). After being treated with RNase‐free DNase I (Roche) and purified by a RNeasy Mini Kit (Qiagen, Valencia, CA, USA), 3 µg of in vitro‐transcribed LINC00662 or LINC00662‐mut was incubated with 1 mg of whole‐cell lysates from HCCLM3 cells for 1 h at 25 °C. Next, the complexes were isolated by streptavidin agarose beads (Invitrogen). The RNA present in the pull‐down material was measured by qRT/PCR.
7
Quantification of circular and linear RHOT1
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RNAs were isolated from HCC samples using TRIZOL (Invitrogen). CircRHOT1, linear RHOT1 and 18S probes for northern blotting were achieved using the Biotin RNA labeling mix (Roche). The RNA samples were separated by electrophoresis and were transferred to NC membranes, which were then incubated with the hydration buffer containing the probes. Finally, the RNA signal was detected using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific). This assay was repeated three times.
8
Biotin-labeled RNA Pulldown for circPTK2
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Biotin-labeled RNA for liner sequence of circPTK2 was generated by an in vitro transcription reaction with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche, Mannheim, Germany), and then treated with RNase-free DNase I (Takara, Japan). After incubation with guide oligonucleotide targeting circular junction, the liner probe was then circularized using T4 RNA ligase I, treated with RNase R. After purified with RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), the biotin-labeled RNA probe (3 μg) was then incubated with cell extracts from CRC cells at room temperature (RT) for 2 h, and treated with 35 μl of Streptavidin C1 magnetic beads (Invitrogen) for 1 h. after washed, the retrieved protein was detected by western blot or mass spectrometry analysis (CapitalBio Technology, Beijing, China).
9
Biotin-labeled miRNA Pulldown Assay
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miR-671-5p and miR-NC were biotinylated using the biotin RNA Labeling Mix (Roche, Indianapolis, IN, USA) and transcribed using T7/SP6 RN polymerase (Roche). Then, biotin-labeled miR-671-5p (bio-miR-671-5p) and bio-miR-NC were treated with RNase free DNase I (Promega) and RNeasy Mini kit (Qiagen, Redwood City, CA, USA). Next, M-280 streptavidin magnetic beads were incubated with bio-miR-671-5p or bio-miR-NC at room temperature for 4 h, and then with A172 and LN229 cell lysates. The RNA binding to bio-miR-671-5p or bio-miR-NC was extracted with TRIzol reagent for RT-qPCR.
10
Biotinylated RNA Pulldown Assay
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miR-122-3p, miR-122-3p-mutant (miR-122-3p Mut) with disrupted base pairing between LINC00665 and miR-122-3p or its negative control (NC) were purchased from GenePharma (Shanghai, China). miRNAs were biotin-labeled using the Biotin RNA Labeling Mix (Roche, Basel, Switzerland) and T7/SP6 RNA polymerase (Roche). Whole cell lysates were mixed and incubated with biotinylated RNAs. The complexes were then incubated with Streptavidin agarose beads (Invitrogen) for 1 h at 37°C. The beads were washed, and RNA level were analyzed by qRT-PCR [29 ].
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