Thermo Fisher Scientific Pierce RNA 3` End Biotinylation Kit

Thermo Fisher Scientific Pierce™ RNA 3' End Biotinylation Kit

The 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.

Features

• Non-radioactive: Incorporates a biotin label enabling detection sensitivity similar to radioactivity when used with chemiluminescent systems
• Fast: RNA labeling in 0.5–2 hours with minimal downstream processing
• Easy to use: Includes RNA ligase and optimized reaction buffer
• Economical: Costs less than purchasing synthetic biotinylated RNA probes
• End-labeled: Minimizes disturbance of RNA secondary structure
• Flexible: Suitable for synthetic or in vitro transcribed RNA probes (22–450 nucleotides)

Kit Contents

This complete kit includes:

• Unlabeled RNA strand (positive control)
• Biotinylated RNA probe standard
• RNase inhibitor
• Glycogen
• Ligation-enhancing reagents

Description

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 enables efficient (>70%) single biotin tagging of RNA probes of diverse secondary structures and lengths.

Typical labeling reactions require 50 pmol RNA and a 20-fold excess of biotinylated nucleotide in a 2-hour reaction at 16°C.

• Short RNA probes with minimal secondary structure can be ligated in 30 minutes at 37°C.
• Large or structurally complex RNA probes (e.g., Let-7, hTR) require longer incubation times.

Optimization of biotinylation efficiency for complex RNA molecules can be achieved by:

• Adjusting RNA to biotinylated cytidine ratio
• Increasing incubation time
• Adding DMSO to denature RNA secondary structure

After labeling, the biotinylated RNA is easily precipitated to remove reaction byproducts. The biotin label enables immobilization or detection with streptavidin for:

• RNA electrophoretic mobility shift assays (EMSA)
• RNA pull-down assays
• Nonradioactive blotting assays (e.g., Northern blotting, miRNA profiling)

Specifications