Home › Cell Biology › RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA
Cell Biology JoVE (Open Access) Citable · DOI

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

DOI: 10.3791/57379-v
What you'll learn
  • ✓ Design and synthesize antisense DNA probes targeting specific lncRNAs
  • ✓ Perform RNA pull-down on fixed cells or tissues using biotinylated probes
  • ✓ Identify and validate RNA targets of lncRNAs via hybridization capture
  • ✓ Analyze enriched RNA samples to characterize lncRNA interactomes
Protocol

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Difficulty
advanced
Total time
~4-6 hours per sample (excluding probe synthesis)
Model organism
Cell culture (unspecified); tissue samples compatible
Biosafety
BSL-1

Steps

1
Crosslink, lyse, and sonicate cells or tissues

Fix cultured cells or tissue samples to preserve RNA-RNA interactions, then lyse and sonicate to generate appropriately sized nucleic acid fragments for hybridization.

▶ 01:29
2
Perform RNA pull-down with antisense probes

Incubate lysate with biotin-labeled antisense DNA oligonucleotide probes specific to the target lncRNA, then capture hybridized complexes using streptavidin-coated magnetic beads.

▶ 04:09
3
Identify and validate lncRNA target interactions

Extract and analyze enriched RNA from pull-down samples to identify and confirm RNA targets co-precipitated with the lncRNA of interest using qRT-PCR or RNA-seq.

▶ 06:33
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