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Cat. No.: 3EBRL-20 (for 20T)

Description

3' End Biotin RNA Labeling Kit—also referred to as the RNA 3' End Biotin Labeling Kit, RNA 3' End Biotinylation Kit, RNA 3' Terminal Biotinylation Kit, or simply RNA Biotin Labeling Kit—is a novel and efficient kit designed for labeling the 3' end of RNA with biotin under mild conditions. The biotin-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 Biotinylation Kit (20160).

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 labeling strategies include:

• Radioactive labels (e.g., ³²P, ³⁵S): high sensitivity and specificity, but costly, hazardous, short-lived, and requiring strict handling.

• Non-radioactive labels:

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

The 3' End Biotin RNA Labeling Kit employs a biotin 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 Desthiobiotin RNA Labeling Kit and the 3' End Cy3 RNA Labeling Kit as complementary options.

This kit provides Biotinylated Cytidine (Bis)phosphate, abbreviated as pCp-Biotin (Biotinylated 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 biotin 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-Biotin (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 essential reagents required for RNA 3' end biotin labeling, including pCp-Biotin, 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 biotin-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-Biotin 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 70%. Users can further improve efficiency by optimizing the RNA-to-pCp-Biotin 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 Desthiobiotin RNA Labeling Kit

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

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