raPOOLs - RNA Capture Probes
The raPOOLs (RNA capture probes) are DNA probes that enable specific and efficient isolation of target RNA bound to nucleic acids or proteins.
Reliability
Reproducible and consistent level of RNA enrichment
Customizable
Budget-friendly solution for customized biotinylated probes
Efficiency
Highly proficient output-to-input ratio and specific RNA capture
Time saving
Why capture RNA 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.
raPOOLs (RNA capture probes) 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).
When to use raPOOLs?
> Large scale isolation of RNAs from cell lysates (long non-coding RNAs & premature/messenger RNAs)
> Co-isolation of interactome to study associated proteins or nucleic acids
RNA Affinity Purification Workflow with raPOOLs
The raPOOL workflow includes four steps:
Step 1: 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
Step 2: raPOOL is added to lysates and hybridizes with the RNA of interest.
Step 3: Addition of streptavidin-coated magnetic beads allow isolation of the raPOOL-bound complex through the high affinity biotin-streptavidin interaction.
Step 4: 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, which was reproduced by both raPOOLs. Enrichment was specific to the targeted RNA.
