Thermo Scientific Pierce RNA 3' End Biotinylation Kit is optimized for labeling the 3-prime terminal end of RNA probes to facilitate their use as probes or targets in EMSA and other methods for studying protein-RNA interactions.
The complete kit contains an unlabeled RNA strand to use as a positive control, a biotinylated-RNA probe standard, RNase inhibitor, glycogen and ligation-enhancing reagents.
- Non-radioactive – incorporates a biotin label which allows detection sensitivity similar to radioactivity when used with chemiluminescent detection systems
- Fast – RNA can be labeled in 0.5-2 hours with minimal downstream processing
- Easy to use – RNA ligase and optimized reaction buffer are included
- Economical – only a fraction of the cost of purchasing synthetic biotinylated RNA probes
- End-labeled – results in minimal disturbance of RNA secondary structure
- Flexible – label synthetic or in vitro transcribed RNA probes ranging from 22–450 nucleotides
The RNA 3’ End Biotinylation Kit uses T4 RNA ligase to conjugate a single nucleotide analog to the 3´ terminus of an RNA strand. The unique biotinylated cytidine (bis)phosphate used in this kit enables T4 RNA ligase to attach a single biotin tag on RNA probes of diverse secondary structure and lengths with > 70% efficiency.
|Reaction scheme for the T4 RNA ligation reaction. 3' end labeling of RNA with T4 RNA ligase requires a 3'-OH (from the desired RNA) and a 3', 5' (bis)phosphate nucleotide. The cytidine (bis)phosphate nucleotide included with the Pierce RNA 3' End Labeling Kit is modified with a biotinylated linker to allow detection or pull-down of labeled RNA probes with avidin proteins conjugated to enzymes or immobilized on solid supports, respectively.
Labeling reactions performed with the RNA 3’ End Biotinylation Kit typically require 50 pmol of RNA and a 20-fold excess of biotinylated nucleotide in a 2 hour reaction at 16°C. Shorter RNA probes with minimal secondary structure can be ligated in 30 minutes at 37°C (e.g., RNA polymerase RNA template). Large or structurally complex RNA probes (e.g., Let-7 and hTR) require longer incubation times. Optimization of biotinylation efficiency for complex RNA molecules is achieved by altering the RNA to biotinylated cytidine ratio, increasing the incubation time or by adding DMSO to the labeling reaction in order to denature the RNA structure.
Following the RNA biotinylation reaction, labeled RNA is easily precipitated to remove reaction byproducts. The biotin label enables the RNA probe to be immobilized or detected with streptavidin in order to study RNA interactions (i.e., RNA electrophoretic mobility shift assays, RNA pull-down assays) or to perform nonradioactive blotting assays (i.e., Northern blotting and miRNA profiling).
|The Thermo Scientific Pierce RNA 3' End Labeling Kit produces RNA probes with similar sensitivity to synthetically labeled RNA. Two RNA probes end-labeled with the Pierce 3' End Biotinylation Kit (RNA pol and IRE RNA) and a synthetic biotinylated RNA probe (Synthetic RNA) were electrophoresed on a 10% acrylamide/8M urea gel, transferred to a nylon membrane, UV crosslinked and detected using the Thermo Scientific Chemiluminescent Detection Module (Part No. 89880). RNA probe concentrations are indicated (nM). The membranes were exposed to X-ray film for 5 seconds.
|RNA labeling application: Northern blot. Let-7 sense and anti-sense RNA were in vitro transcribed using T7 (sense) and T3 (anti-sense) RNA polymerase. The sense RNA was electrophoresed on a 5% acrylamide/8M urea denaturing gel, transferred to a nylon membrane, and UV cross-linked. Twenty-five pmol of anti-sense RNA was end-labeled using the Thermo Scientific Pierce RNA 3’ End Biotinylation kit. After an overnight incubation at 16°C, RNA was chloroform:isoamyl alcohol extracted, and ethanol precipitated. Ligation efficiency was assessed using a dot blot. Ten pmol of RNA probe was used for northern analysis using the Thermo Scientific North2South Hybridization and Detection Kit according to instructions. Concentrations are indicated and the exposure time was 2 minutes.
||RNA probes biotinylated with the Thermo Scientific Pierce RNA 3' End Biotinylation Kit retain function and interact with RNA binding proteins. Iron response element (IRE), RNA Polymerase, and Let-7 RNA probes were biotinylated using the Pierce RNA 3' End Biotinylation Kit. Each biotinylated RNA probe (5-10nM) was tested for functionality in a RNA binding reaction with 4-5µg of cell extract using the Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit (Product # 20158) to detect interactions with Iron-responsive Protein (IRP), RNA Polymerase or Lin28. Lane 1: free probe; Lane 2: binding reaction; Lane 3: binding reaction plus 100-fold excess of unlabeled probe. The presence of shifted bands (arrows) in lane 2 of each experiment shows RNA-protein complexes in the binding reactions, which are competed away by the addition of excess unlabeled RNA probe (Lane 3).
- Hall-Pogar, T., et al. (2007). Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3'-UTR. RNA 13:1103-15.
- Leibold, E.A. and Munro, H.M. (1988). Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl Acad Sci USA 85:2171-5.
- 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.
- O’Connor, C.M., et al. (2005). Two purified domains of telomerase reverse transcriptase reconstitute sequence-specific interactions with RNA. J Biol Chem 280:17533-9.
- Piskounova, E., et al. (2008). Determinants of microRNA processing by the developmentally regulated RNA binding protein Lin28. J Biol Chem 283:21310-4.
- Sukhodolets, M.V. and Jin, D.J. (1998). RapA, a novel RNA polymerase-associated protein, is a bacterial homology of SWI2/SNF2. J Biol Chem 273:7018-23.
- Ueda, C.T. and Roberts, R.W. (2004). Analysis of a long range interaction between conserved domains of human telomerase RNA. RNA 10:139-47.
Protein Methods Library: Protein-Nucleic Acid Interactions Overview
Protein Methods Library: Methods for Detecting Protein-RNA Interactions
Biotinylation Reagent Selection Guide
Protein-Nucleic Acid Interactions Technical Handbook
LightShift RNA EMSA Kit
Chemiluminescent Nucleic Acid Detection Module and Blocking Buffer