Robust & Targeted RNA Capture with raPOOLs

The discovery that 98% of the human genome is non-(protein) coding has fueled extensive interest in the functions of non-coding RNA (ncRNA). Long non-coding RNAs (lncRNAs), have been implicated in various physiological processes and disease pathogenesis. As lncRNAs interact with protein, DNA and RNA to exert their effects, biochemical interaction studies to resolve their binding partners can provide valuable insight into their function.

RNA capture probes (raPOOLs) consist of 30 optimally-designed 3'-biotinylated DNA probes that enable specific and efficient isolation of target RNA bound to nucleic acids or proteins. Used with streptavidin-coated magnetic beads, raPOOLs have shown robust RNA enrichment and were published to successfully isolate known and novel lncRNA-interacting proteins (Nötzold et al. 2017).

Benefits of using raPOOLs

RNA Affinity Purification with raPOOLs: How It Works

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).

Supporting Data: Targeted, robust RNA capture with raPOOLs

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.