The template for tRSA-RNAIII transcription was digested from plasmid pSXZ06. The RNAs were produced by in vitro transcription as described [36 (link)], using a RiboMAX Large Scale RNA Production Systems-T7 (Promega). RNAs were labeled with DIG using RNA labeling Mix (Roche).
Rna labeling mix
Manufactured by Roche
Sourced in United States
The RNA Labeling Mix is a reagent used for the labeling of RNA molecules. It contains the necessary components for the incorporation of labeled nucleotides into RNA samples, enabling the detection and analysis of RNA transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Anti dig antibody, by Roche (2 mentions)
Ribomax large scale rna production systems t7, by Promega (1 mentions)
T3 rna polymerase, by Roche (1 mentions)
Anti dig pod, by Roche (1 mentions)
Anti dig ap, by Roche (1 mentions)
Nbt bcip solution, by Roche (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Nucleospin rna isolation kit, by Macherey-Nagel (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
T3 t7 rna polymerase, by Roche (1 mentions)
Mascot daemon software, by Matrix Science (1 mentions)
Digital sight ds fi1 camera, by Nikon (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
T3 or t7 rna polymerase, by Roche (1 mentions)
12 protocols using rna labeling mix
1
In Vitro Transcription of tRSA-RNAIII
2
Biotin-labeled Cpmer RNA Purification
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Biotin-labeled Cpmer was obtained using RNA Labeling Mix (11685597910, Roche) with T7 (10881767001, Roche) or T3 RNA polymerase (11031171001, Roche). Total RNA was heated to 90°C for 2 min, held on ice for 2 min, and incubated in RNA structure buffer. Lysis was performed using streptavidin beads coated with biotin-labeled sense Cpmer or antisense Cpmer. The RNA-binding proteins were analyzed by LC-MS as described previously (Shevchenko et al., 2006 (link)).
3
Affinity Purification of RNA-Binding Proteins
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Briefly, biotin-labeled RNAs were obtained using RNA Labeling Mix (Roche, 11685597910) and T7/T3 RNA polymerase (Roche, 10881767001/11031171001). A total of 3 μg of the RNA was heated to 90°C for 2 min, held on ice for 2 min in RNA structure buffer (10 mM Tris-HCl pH 7.0, 0.1 M KCl, and 10 mM MgCl2) to allow proper secondary structure formation. ESCs (5 × 106 cells) were lysed with RIP lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM Hepes pH 7.0, and 0.5% NP-40) for 30 min on ice to facilitate lysis. Immunoprecipitation was subsequently performed using streptavidin beads coated with 3 μg of the biotin-labeled RNAs, according to the manufacturer’s instructions. The RNA-binding proteins were treated with SDS lysis buffer for subsequent western blotting analysis.
4
Whole-mount and Sectional FISH Protocols
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Whole-mount chromogenic in situ hybridization and whole-mount fluorescent in situ hybridization (FISH) was performed as detailed by Marchal and colleagues54 (link), and Castillo-Briceno and Kodjabachian55 (link), respectively. For single staining, all RNA probes were labeled with digoxigenin. For FISH on section, embryos were fixed in 4% paraformaldehyde (PFA), stored in methanol for at least 4 h at −20 °C, then rehydrated in PBT (PBS + Tween 0.1% v/v), treated with triethanolamine and acetic anhydride, incubated in increasing sucrose concentrations and finally embedded with OCT (VWR Chemicals). 12 μm-thick cryosections were made. Double FISH on sections was an adaptation of the whole-mount FISH method. 80 ng of cdc20b digoxigenin-labeled sense and antisense riboprobes and 40 ng of antisense α-tubulin fluorescein-labeled riboprobe56 (link) were used for hybridization. All probes were generated from linearized plasmids using RNA-labeling mix (Roche). FISH was carried out using Tyramide Signal Amplification – TSA TM Plus Cyanine 3/Fluorescein System (Perkin Elmer). Antibodies: Anti-DigAP (Roche, 11266026, 1:5000), Anti-DigPOD (Roche, 11207733910, 1:500), Anti-FluoPOD (Roche, 11426346910, 1:500).
5
Antisense RNA Probe Synthesis Protocol
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The templates for anti-sense RNA probe synthesis were produced by PCR from embryonic cDNA.
All primers for probe synthesis are listed in S1 Table. The T7 promoter sequence was added to the 5'-end of anti-sense primers. The RNA probes were synthesized using T7 RNA polymerase with DIG under 17 USC 105 and is also made available for use under a CC0 license. preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright
The copyright holder for this this version posted December 7, 2022. ; https://doi.org/10.1101/2022.12.05.519245 doi: bioRxiv preprint RNA Labeling Mix (Roche). The larvae were fixed in 4% formaldehyde at 4°C overnight, dehydrated with 100% methanol and stored at -20°C. Samples were rehydrated into PBST (1xPBS with 0.1% tween-20) gradually, bleached with 10% H 2 O 2 , permeabilized with 10 μg/μL proteinase K for 10 min, then post-fixed in 4% PFA for 20 min. Hybridization was carried out at 70°C overnight, followed by post-hybridization washing, blocking with 2% bovine serum albumin (BSA) and 2% sheep serum, incubation with anti-DIG antibody (Roche), and stained with NBT/BCIP solution (Roche).
All primers for probe synthesis are listed in S1 Table. The T7 promoter sequence was added to the 5'-end of anti-sense primers. The RNA probes were synthesized using T7 RNA polymerase with DIG under 17 USC 105 and is also made available for use under a CC0 license. preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright
The copyright holder for this this version posted December 7, 2022. ; https://doi.org/10.1101/2022.12.05.519245 doi: bioRxiv preprint RNA Labeling Mix (Roche). The larvae were fixed in 4% formaldehyde at 4°C overnight, dehydrated with 100% methanol and stored at -20°C. Samples were rehydrated into PBST (1xPBS with 0.1% tween-20) gradually, bleached with 10% H 2 O 2 , permeabilized with 10 μg/μL proteinase K for 10 min, then post-fixed in 4% PFA for 20 min. Hybridization was carried out at 70°C overnight, followed by post-hybridization washing, blocking with 2% bovine serum albumin (BSA) and 2% sheep serum, incubation with anti-DIG antibody (Roche), and stained with NBT/BCIP solution (Roche).
6
Biotin-labeled HHIP-AS1 RNA Pulldown
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Bio (biotinylated)-NC, bio-HHIP-AS1 OL, bio-HHIP-AS1 non-OL labelled with RNA Labeling Mix (Roche, Mannheim, Germany) in vitro were transfected into BMSCs. The cells were lysed for 1h and collected using streptavidin-coupled magnetic bead (Invitrogen). The protein-bio/RNA-magnetic bead complex was isolated using high salt elution. The protein-bio/RNA was extracted and purified with TRIzol Kit, followed by qRT-PCR of HHIP mRNA.
7
Fluorescent In Situ Hybridization Protocol
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Sections were prepared for fluorescent in situ hybridization using the same probes as those from the in situ hybridization experiments, with the exception that FITC (CRF) and DIG (Grin1), RNA labeling mix (Roche) was used instead of S35 as previously described [26] (link). Slides were hybridized at 65°C for at least 12 h, followed by a series of stringent washes. Sections were then blocked with 1% TNB buffer (1% BSA in TN) for 30 min, treated with peroxidase followed by anti-DIG antibody (1:500 dilution; Roche) for 1.5 h. Sections were rinsed and treated with Cy3 antibody (1:50 dilution; Roche) for 30 min in the dark. After further rinses, sections were similarly treated with anti FITC antibody (1:500 dilution; Vector Laboratories) followed by FITC tyramide (1:50) for amplification in a humid chamber in the dark. Finally, sections were stained with Hoescht stain and cover-slipped with Vectashield (Vector Laboratories).
8
BCRP3 RNA-protein Interaction Analysis
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The full-length BCRP3 cDNA was cloned to pcDNA3.1 in a sense direction. Biotin-labeled BCRP3 was synthesized by in vitro transcription using RNA Labeling Mix (Roche) and T7 RNA polymerase (Ambion, Austin, TX, USA), and purified by NucleoSpin® RNA Isolation kit (Macherey–Nagel, Bethlehem, PA, USA). Biotinylated BCRP3 was folded in RNA structure buffer (10 mM Tris–HCl pH 7.0, 100 mM KCl, and 10 mM MgCl2) for 2 min at 90℃, immediately chilled on ice for 2 min, and incubated at room temperature for 20 min to form the proper secondary structure. GFP-ATG14 plasmid was transfected into 293T cells and 1.5 mg cell lysates were immunoprecipitated with 80 μl 50% slurry of GFP-TRAP beads. The ATG14 immunocomplexes were then incubated with in vitro synthesized folded BCRP3 (2 pmol) for 1 h at 4℃. After washes, the beads were divided into two aliquots. One of the aliquots was used for Western blotting while the other was subjected to RNA extraction with Trizol reagent, followed by qRT-PCR analysis.
9
Identifying linc1281-binding proteins in mESCs
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To identify linc1281-interacting proteins in mESCs, biotin-RNA pull down and subsequent liquid chromatography-mass spectrometry were performed. Briefly, biotin-labeled RNAs were synthesized by using RNA Labeling Mix (Roche, 11685597910) and T3/T7 RNA polymerase (Roche, 10881767001/11031171001). Linc1281 or antisense RNAs were heated to 90°C for 2 min, held on ice for 2 min and then incubated in room temperature for 20 min in RNA structure buffer (10 mM Tris–HCl pH 7.0, 0.1 M KCl and 10 mM MgCl2) to form proper secondary structure. mESCs (1 × 107 cells) were lysed with RIP lysis buffer and Immunoprecipitation was performed by using streptavidin beads coated with 3 μg of linc1281 or antisense RNAs. The RNA-binding proteins were sequenced and identified by LC–MS as described previously (41 (link)). The mass spectra were searched using the Mascot Daemon software (Version 2.3.0, Matrix Science, London, UK) based on the Mascot algorithm. The database used to search was the Mouse UniProtKB database.
10
Whole-mount in situ hybridization of Xenopus laevis
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All experiments were performed following the Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes and approved by the "Direction départementale de la Protection des Populations, Pôle Alimentation, Santé Animale, Environnement, des Bouches du Rhône " (agreement number F1305521).
RNA probes and whole mount in situ hybridization cDNA fragments from Xenopus laevis lrrcc1.L (entrez gene 431936), and ccdc61.L (entrez gene 734655) were amplified from commercial cDNA (Horizon discovery) by PCR using the following primers: lrrcc1 forward: 5'-GCGAACGGACACAGACAGTA-3'; lrrcc1 reverse: 5'-GAATTCCATGGTAGTCAGCTCCTGC-3'; ccdc61 forward: 5'-GCGGCCGCAAGTGGAGGATGCTGTGACC-3'; ccdc61 reverse: 5'-GAATTCACGGATGAACTGCGTCTCTG-3'.
PCR products were cloned in pBlueScript KS+ vector and digoxigenin-labelled probes were generated from linearized plasmids using RNA-labeling mix (Roche). Whole-mount chromogenic in situ hybridization was performed as described previously (Marchal 2009) using 40ng of Digoxygenin-labelled probe. Pictures were taken with the stereomicroscope Leica MZ125 coupled to NIKON digital Sight DS-Fi1 camera.
RNA probes and whole mount in situ hybridization cDNA fragments from Xenopus laevis lrrcc1.L (entrez gene 431936), and ccdc61.L (entrez gene 734655) were amplified from commercial cDNA (Horizon discovery) by PCR using the following primers: lrrcc1 forward: 5'-GCGAACGGACACAGACAGTA-3'; lrrcc1 reverse: 5'-GAATTCCATGGTAGTCAGCTCCTGC-3'; ccdc61 forward: 5'-GCGGCCGCAAGTGGAGGATGCTGTGACC-3'; ccdc61 reverse: 5'-GAATTCACGGATGAACTGCGTCTCTG-3'.
PCR products were cloned in pBlueScript KS+ vector and digoxigenin-labelled probes were generated from linearized plasmids using RNA-labeling mix (Roche). Whole-mount chromogenic in situ hybridization was performed as described previously (Marchal 2009) using 40ng of Digoxygenin-labelled probe. Pictures were taken with the stereomicroscope Leica MZ125 coupled to NIKON digital Sight DS-Fi1 camera.
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