Biopharma Insights 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.
Total time
~4–6 hours per sample (including crosslinking, extract preparation, and immunoblot detection)
Steps
1
Design antisense oligonucleotides for target mRNA
Design 2′-methoxy-modified antisense RNA oligonucleotides complementary to target mRNA sequences. Synthesize with 3′ biotin modification for downstream capture and detection.
▶ 00:32
2
Prepare protein extracts from crosslinked cells
UV-crosslink cells in vivo to stabilize RNA-protein interactions, then lyse and prepare whole-cell extracts for downstream isolation.
▶ 02:22
3
Isolate polyadenylated RNA with oligo(dT) beads
Incubate cell extracts with oligo(dT)-coated magnetic beads to capture all poly(A) RNA, then wash and elute enriched RNA-protein complexes.
▶ 04:08
4
Capture specific mRNA with biotinylated antisense oligonucleotides
Add 3′-biotinylated 2′-methoxy-modified antisense oligonucleotides to isolated poly(A) RNA to specifically pull down target mRNA-protein complexes via streptavidin interaction.
▶ 05:45
5
Detect bound proteins by immunoblot analysis
Isolate captured mRNA-protein complexes and perform immunoblot with target protein antibodies to identify proteins crosslinked to specific mRNAs.
▶ 07:41