Firstly, TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) was used to generate the full length fragments of PVT1 and anti-sense-PVT1. Then PVT1 and anti-sense-PVT1 were labeled with Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific). RNA-protein pull-down assays were carried out using Pierce Magnetic RNA-Protein Pull down Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Briefly, labeled RNA bound with Streptavidin Magnetic Beads, followed by the binding between RNA and RNA-binding proteins from MCF-7 cells lysate. Then the RNA-protein complexes were eluted for the further western blot analysis.
Thermo scientific pierce rna 3 desthiobiotinylation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Scientific Pierce RNA 3' Desthiobiotinylation Kit is a tool for labeling the 3' end of RNA molecules with a desthiobiotin tag. This kit provides the necessary reagents and protocols to perform this 3' end-labeling procedure.
Automatically generated - may contain errors
4 protocols using thermo scientific pierce rna 3 desthiobiotinylation kit
1
PVT1 RNA-Protein Interactome Identification
2
RNA-Protein Interactome Analysis
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TUG1 and anti-sense-TUG1 was transcribed with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and then labeled with Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific). Pierce Magnetic RNA-Protein Pull down Kit (Thermo Fisher Scientific) was used to perform RNA pull down assay. Briefly, labeled RNAs were bound with Streptavidin Magnetic Beads and then incubated with ECA109/DDP cell protein lysates. Then the RNA-binding proteins were eluted for the further western blot analysis.
3
In Vitro Transcription and Protein Pulldown
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In vitro transcription was performed as described previously [43 (link)]. Briefly, the pCDNA3.1-AOC4P plasmid containing a T7 promoter was linearized via BamHI digestion, purified, and used as a template for in vitro transcription. The template was incubated in 1 mM each of ATP, CTP, GTP, UTP and T7 RNA polymerase in 1 × transcription buffer. The in vitro-transcribed RNA was purified using an RNeasy purification kit (Qiagen, Germany). The transcripts were labeled with biotin using a Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific, MA, USA). The RNA-protein binding reaction was performed using freshly harvested SK-Hep1 cells and the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, MA, USA). The reaction products were then subjected to mass spectrometry for protein identification.
4
In vitro Transcription and RNA-Protein Binding
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In vitro transcription was performed as previously described [46 (link)]. Briefly, the pCDNA3.1-CPS1-IT1 plasmid containing a T7 promoter was linearized by BamHI digestion, purified, and used as a template for in vitro transcription. The template was incubated in ATP, CTP, GTP, and UTP (1 mM each) plus T7 RNA polymerase in 1× transcription buffer. The in vitro-transcribed RNA was purified using an RNeasy purification kit (Qiagen, Germany), and the transcripts were labeled with biotin using a Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific, MA, USA). The RNA-protein binding reaction was performed using freshly harvested SK-Hep1 cells and a Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, MA, USA). The reaction products were then subjected to mass spectrometry for protein identification.
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