The Thermo Scientific Pierce RNA 3’-End Desthiobiotinylation Kit contains reagents for easy and efficient desthiobiotin labeling of RNA for use as probes or as antibody alternatives for enrichment of RNA binding proteins.
The Pierce RNA 3'-End Desthiobiotinylation Kit is optimized for labeling the 3'-end of single-stranded RNA using T4 RNA ligase. Once labeled, the RNA can be used as a probe or target for gel-shift EMSA reactions, Northern blots and protein-RNA interaction experiments. The desthiobiotinylated RNA also can be used to enrich for RNA binding proteins (RBP) using streptavidin affinity resin, because the desthiobiotin tag binds to streptavidin in a manner which allows gentle elution of the ribonucleoprotein complex. The labeling kit is also included as a component of the Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit.
- Non-radioactive – a single biotin labels has similar detection sensitivity as radioactivity
- Easy to use – RNA ligase and optimized reaction buffer are included
- Economical – only a fraction of the cost purchasing synthetic biotinylated RNA probes
- End-labeled – results in minimal disturbance of RNA secondary structure
- Flexible – desthiobiotin label may be used for detection or as an affinity handle for streptavidin resin
Desthiobiotinylated cytidine bisphosphate, T4 RNA ligase and buffer, unlabeled RNA oligonucleotide for use as a positive control, a biotinylated RNA probe standard, RNase inhibitor, glycogen, and ligation enhancing reagents
- Northern blotting
- Enrichment of RNA binding proteins (RBP)
This RNA labeling kit uses T4 RNA ligase to attach a single desthiobiotinylated cytidine bisphosphate to the 3-’end of single-stranded RNA. Each labeling reaction was designed for 50pmol of RNA; however, reactions may be scaled (1pmol to 1nmol have been tested), if necessary. The reaction uses a 20-fold excess of desthiobiotinylated nucleotide and requires incubation times from 30 minutes at 37°C (for less complex RNA) to overnight at 4 to 16°C (for longer or more complex RNA). Optimization of the labeling efficiency for complex RNA structures is achieved by altering the RNA:nucleotide ratio, increasing the incubation time, or by adding DMSO to relax the RNA structure. After organic extraction and precipitation with ethanol, the desthiobiotin-labeled RNA is ready for use in downstream applications.
|Reaction scheme for the T4 RNA ligation reaction. Note that the desthiobiotin can be used for detection or for enrichment using streptavidin conjugates or affinity supports, respectively.
Example experiments with different RNA molecules demonstrate the robustness of the ligation reaction. Synthetic RNA and in vitro transcribed RNA were evaluated, and ligations efficiencies were determined by semi-quantitative dot blot using a synthetic biotinylated RNA as the 100% labeled control (Table). Ligation efficiencies were all greater than 50%, with many greater than 70%. The kit control RNA had greater than 90% efficiency, and serves as an indicator of the overall reaction conditions and reagents. Further optimization was necessary for RNA having significant secondary structure, and refolding was required for in vitro transcribed RNA after ligation.
Ligation efficiency of RNA desthiobiotinylation. RNA was heated at 90°C for five minutes and quick-chilled before ligation. Each reaction contained 50pmol of RNA, and ligation reactions were performed according to the product instructions. Relative ligation efficiencies were determined by dot blot using densitometry and compared to a synthetic biotinylated RNA control (100%). Each ligation was performed in duplicate, and spotted in triplicate.
A pull-down assay involving the let-7 miRNA:Lin28 interaction demonstrates the experimental functionality of desthiobiotinylated-labeled RNA. The developmentally regulated Lin28 protein is a selective inhibitor of let-7 miRNA processing through binding to a loop region of let-7 pre-miRNA. To test this interaction, the loop portion of let-7 miRNA was end-labeled with desthiobiotinylated cytidine and attached to streptavidin magnetic beads. Beads were subsequently incubated with embryonal carcinoma cell lysate (NCCIT) in Protein-RNA binding buffer. Samples were eluted and assayed by Western blotting. The results indicate that the labeled let-7 miRNA specifically enriched Lin28 protein from NCCIT lysate, while an unrelated RNA did not.
|Let-7 loop RNA specifically enriches Lin28 in NCCIT extract. Desthiobiotinylated let-7 miRNA (50 pmol) was used to enrich Lin28 using the Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit (Part No. 20164), followed by Western blotting. Samples were normalized by volume. L – lysate load; FT – flow-through, E – eluate. Unrelated – negative control poly(A)25 RNA. Anti-Lin28 antibody (Coming Soon!).
This kit is also included in The Pierce Magnetic RNA-Protein Pull-Down Kit and utilizes the labeling kit in conjunction with optimized binding, wash, and elution buffers to enrich RNA binding proteins.
- England, T.E., et al. (1980). Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzym 65:65-74.
- Keith, G. (1983). Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase. Biochimie 65:367-70.
- McKinley, B.A. and Sukhodolets, M.V. (2007). Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity. Nucleic Acids Res 35:7044-60.
- Khanam, T., et al. (2006). Poly(A)-binding protein binds to A-rich sequences via RNA-binding domains 1+2 and 3+4. RNA Biology 3: 170-7.
- Piskounova, E., et al. (2008). Determinants of micoRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J. Biol. Chem. 283: 21310-4.
Review of protein-nucleic acid interactions
Methods for detecting protein-RNA interactions
Methods for labeling nucleic acids
Magnetic RNA-Protein Pull-Down Kit
Pierce RNA 3' End Biotinylation Kit
LightShift Chemiluminescent RNA EMSA Kit
Other Nucleic Acid Labeling and Detection Products
Chromatin IP Kit (ChIP)