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Cat. No.: 3EDRL-20 (for 20T)

Description

3' End Desthiobiotin RNA Labeling Kit—also referred to as the RNA 3' End Desthiobiotin Labeling Kit, RNA 3' End Desthiobiotinylation Kit, RNA 3' Terminal Desthiobiotinylation Kit, or simply RNA Desthiobiotin Labeling Kit—is a novel and efficient kit designed for labeling the 3' end of RNA with desthiobiotin under mild conditions. The desthiobiotin-labeled RNA prepared with this kit is suitable for studies of RNA–protein, RNA–RNA, and RNA–DNA interactions such as pull-down assays and EMSA, as well as for applications including Northern blotting, RNA blotting, in situ hybridization, and miRNA profiling.

This kit operates on principles and methods essentially consistent with Thermo’s Pierce™ RNA 3' End Desthiobiotinylation Kit (20163).

RNA-binding proteins (RBPs) regulate RNA processing, maturation, transport, stability, translation, and degradation, thereby supporting cell survival and growth. Investigating RNA–protein interactions is critical for understanding normal cellular function and regulation. RBPs are difficult to isolate independently and are highly dependent on RNA secondary structure, which often necessitates RNA labeling. Common RNA labels include radioactive and non-radioactive markers.

• Radioactive isotopes (e.g., ³²P, ³⁵S) offer high sensitivity and specificity but require strict handling, are costly, have short half-lives, and pose health risks.

• Non-radioactive labels include:

  • Digoxigenin (DIG): high sensitivity, low nonspecific binding, widely used, though labeling is relatively complex.

  • Fluorophores (FITC, PE, Cy3, Cy5, etc.): convenient detection, but fluorescence is prone to quenching and has application limitations.

  • Biotin/desthiobiotin: high sensitivity and specificity, simple labeling, and easy downstream detection.

The 3' End Desthiobiotin RNA Labeling Kit employs a desthiobiotin end-labeling strategy that minimally affects RNA secondary structure and typically does not interfere with RNA–protein interactions, making it one of the preferred non-radioactive RNA labeling solutions. We also offer the 3' End Biotin RNA Labeling Kit and the 3' End Cy3 RNA Labeling Kit as alternatives.

Desthiobiotin is a monocyclic, sulfur-free analog of biotin. Compared with biotin, it binds streptavidin with nearly the same specificity but with lower affinity (Kd ≈ 10⁻¹¹ vs. 10⁻¹⁵ M). This allows desthiobiotin-labeled molecules to be competitively displaced from streptavidin under mild conditions using biotin-containing elution buffers.

This kit provides Desthiobiotinylated Cytidine (Bis)phosphate, abbreviated as pCp-Desthiobiotin (Desthiobiotinylated Cytidine 3',5'-Bisphosphate). Its unique structure features a phosphate group at the 5' position of the ribose ring (5'-P), which can be ligated to the 3'-hydroxyl (-OH) of RNA by T4 RNA Ligase. Meanwhile, the 3'-phosphate group (3'-P) carries a desthiobiotin moiety for subsequent detection. After labeling, the RNA can be purified by chloroform/isoamyl alcohol extraction and isopropanol precipitation, effectively removing reaction by-products.

Components

• pCp-Desthiobiotin (1mM)
• T4 RNA Ligase (10U/μl)
• Reaction Buffer (10X)
• ATP (10mM)
• PEG8000 Solution (RNase-free)
• RNase Inhibitor (40U/μl)
• Non-labeled RNA Control (10μM)
• Biotinylated RNA Control (1μM)
• Glycogen (20mg/ml)
• Ultrapure Water

Features

• Comprehensive Components and High Cost-Performance This kit provides all the necessary reagents for RNA 3' end desthiobiotin labeling, including pCp-Desthiobiotin, T4 RNA Ligase, Reaction Buffer, RNase Inhibitor, and Ultrapure Water. It also includes useful controls such as a Non-labeled RNA Control and a Biotinylated RNA Control. Compared with purchasing pre-synthesized desthiobiotin-labeled RNA probes, this kit is more economical and practical, offering superior cost-effectiveness.
• Non-Radioactive and Flexible Labeling The kit enables efficient, non-radioactive labeling of hydroxyl groups at the RNA 3' end. It is commonly used for labeling synthetic or in vitro transcribed RNA in the range of 10–500 nucleotides.
• Fast Labeling and High Efficiency Under standard conditions, labeling can be achieved by incubating 50 pmol RNA with a 20-fold molar excess of pCp-Desthiobiotin at 25 °C for 2 hours. For RNA probes with weaker secondary structures, the reaction can be completed in as little as 30 minutes at 37 °C. For RNA with strong or complex secondary structures, the reaction time may need to be extended. Testing has shown that the labeling efficiency of this kit exceeds 90%. Users can further improve efficiency by optimizing the RNA-to-pCp-Desthiobiotin ratio or by adding DMSO to weaken RNA secondary structures during the reaction.

Storage 

Store at –20 °C. Stable for one year.

Notes

• All reagents provided in this kit are RNase-free. Handle with care to avoid contamination.
• All user-prepared reagents and consumables must also be RNase-free. If consumables may be contaminated with RNase, they can be soaked overnight in 0.01% DEPC-treated water, followed by autoclaving and drying.
• Avoid breathing, talking, or exhaling directly over samples or kit components to prevent RNase contamination. Wearing a disposable mask during operation is recommended.
• Ensure that the RNA to be labeled has a 3' hydroxyl group.
• This product is intended for scientific research use only by trained professionals. It must not be used for clinical diagnosis or therapy, in food or pharmaceuticals, or stored in residential settings.
• For your safety and health, always wear a lab coat and disposable gloves when handling this product.

Related: 3' End Biotin RNA Labeling Kit

Only for research and not intended for treatment of humans or animals

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