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An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes
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Biochimie
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Journal JoVE Biochimie
An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

DOI:
10.3791/58223-v

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09:45 min

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August 18, 2018

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, ,
1Dept. of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences,University of Surrey

Chapitres

  • 00:04Titre
  • 00:32Antisense Oligonucleotide (ASO) Design
  • 02:22Protein Extract Preparation
  • 04:08Step 1: Poly(A) RNA Isolation
  • 05:45Step 2: Specific mRNA Capture with 3 -Biotinylated 2 -Methoxy Modified Antisense RNA Oligonucleotides
  • 07:41Results: Capture of Specific mRNA-protein Complexes from Extracts Derived from UV Crosslinked Cells with TRIP
  • 09:00Conclusion

Summary

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A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

Tags

Keywords: OligonucleotideTandem RNA IsolationMRNA-protein ComplexesGene Expression RegulationRNA-binding ProteinsPost-transcriptional Gene RegulationEukaryotic MRNASecondary Structure AnalysisBiotinylated OligonucleotidesHEK293 CellsUV Crosslinking

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