RNA Affinity Purification with raPOOLs
Nucleic acid and protein interactions are preserved with a cross-linking reagent, followed by lysis and sonication to shear nucleic acids to sizes amenable for pulldown. The raPOOL is added to lysates and hybridizes with the RNA of interest. Addition of streptavidin-coated magnetic beads allow isolation of the raPOOL-bound complex through the high affinity biotin-streptavidin interaction. The complexes are then disrupted and individual components analysed by various methods: western blotting/mass spectrometry (for proteins), sequencing/northern and southern blots/PCR detection (for nucleic acids).
Workflow
The raPOOL workflow includes three steps:
Step 1. raPOOL is added to lysates and hybridizes with the RNA of interest.
Step 2. Addition of streptavidin-coated magnetic beads allow isolation of the raPOOL-bound complex through the high affinity biotin-streptavidin interaction.
Step 3. The complexes are then disrupted and individual components analysed by various methods: western blotting/mass spectrometry (for proteins), sequencing/northern and southern blots/PCR detection (for nucleic acids).
raPOOL Performance
Lysates from HeLa cells were incubated with two different raPOOLs against human MALAT1, human XIST or E. coli LacZ (negative control) for 4 h at 37°C. Following RNA isolation and cDNA synthesis, levels of Cyclophilin A (PPIA), GAPDH or targeted lncRNA, MALAT1, were measured by real-time quantitative PCR. Approximately 100-fold RNA enrichment of the target (MALAT1) was detected that was reproduced by both raPOOLs. Enrichment was specific to the targeted RNA.
