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Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs
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Genetics
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JoVE Journal Genetics
Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

DOI:
10.3791/57786-v

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11:00 min

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June 12, 2018

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, , ,
1School of Biotechnology,Kalinga Institute of Industrial Technology University, 21. Center for AIDS Health Disparities Research Department of Biochemistry and Cancer Biology,Meharry Medical College, 3Kalinga Institute of Industrial Technology University

Chapters

  • 00:04Title
  • 01:34Preparation of Streptavidin Coated Magnetic Beads — I
  • 03:52Preparation of Cell Lysate
  • 05:36Preparation of Streptavidin Magnetic Beads — II
  • 06:34Pull-down of Target mRNA-miRNA Complexes
  • 07:35Total RNA Extracted, cDNA Synthesis, and qPCR
  • 08:57Results: Use of MicroRNAs to Capture the Target mRNA
  • 09:55Conclusion

Summary

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This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

Tags

Keywords: Biotinylated MiRNA MimicsMRNA TargetsPulldown AssayLocked Nucleic AcidsStreptavidin-coated Magnetic BeadsMiRNA-mRNA InteractionsMiRNA BiologyMiRNA Therapeutics

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