raPOOLs - RNA Capture Probes
raPOOLs (RNA antisense pools) are biotinilated DNA probes that enable specific and efficient isolation of target RNA bound to nucleic acids or proteins from cell lysates.
Reliability
Reproducible and consistent level of RNA enrichment
Customizable
Budget-friendly solution for customized biotinylated probes
Efficiency
highly efficient and specific targeting
HPLC purified
higher purity for cleaner hybridization signals
Principle
raPOOLs (RNA antisense pools) enable robust, specific isolation of targeted RNA from cell lysates.They are highly complex sets of biotinylated DNA probes, each consisting of 30 optimally designed 3’-biotinylated sequences. Their high complexity pooling provides maximum coverage of the target RNA for strong enrichment efficiency. The workflow is compatible with cross-linked samples, allowing isolation as well as identification and characterization of nucleic acids and proteins interacting with the target RNA.
Applications
> 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).
lncRNA Interactome Analysis Using 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. 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).
raPOOL Performance
Use Case 1: Enrichment of human MALAT1
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.
Figure 1: Real-time qPCR quantification of MALAT1 (targeted RNA), PPIA (control) and GAPDH (control) RNA
in Hela S3 cell lines after pull down with MALAT1, XIST and negatice control raPOOLs.
Use Case 2: lncRNA interactome
A siRNA screen targeting lncRNAs deregulated in cancer, found LINC00152, a lncRNA with upregulated expression levels in different cancer types, to affect cell morphology
and cell division. Pulldown of LINC00152 in Hela cells and identification of its associated proteins by mass spectrometry, revealed that LINC00152 interacts with a network of proteins associated with the M phase of the cell cycle. This suggests that this lncRNA is crucial for cell cycle progression through mitosis and thus, could act as a non-codig oncogene.
Nötzold et all, Scientifc Reports, 2017
Figure 2: Interaction networks between identified proteins associated with M phase as revealed by the String database.
Proteins had a function in microtubule cytoskeleton organization (green nodes) and ubiquitin-protein ligase activity (cyan nodes),
with cyclin B (CCNB1) being involved in both (blue).
Use Case 3: Ribosome Heterogeneity
A new highly-divergent type (F-type) of rRNA was identified, that is expressed during the early stages of parasite development in malaria mosquitos.
A custom raPOOL probe set was produced to specifically pull-down down F-type rRNA from P.falciparim cell lysates.
Initial validation via qPCR showed a > 30x enrichment of F1 rRNA after pull-down, confirming raPOOL efficiency.
Sebastian Baumgarten, Parasite RNA Biology lab, Institut Pasteur, Paris,France
Figure 3: Expression of F-type 28S rRNA (F1) in cell lysates before and after raPOOL enrichment.
Reference gene for normalization: seryl-tRNA synthetase..