• 5' end label and purify small RNA and DNA probes
  
  
• Includes 10 – 150 nt Decade™ Markers – ideal size markers for gel analysis of small RNAs
  
  
• Simple and rapid post-labeling probe clean up procedure
  
  
• Ideal for small RNA analysis by Northern blot or with the mirVana™ miRNA Detection Kit
  
  
• Compatible with chemically synthesized oligonucleotides -- no need to gel purify after labeling
  
  

The mirVana Probe & Marker Kit is designed for rapid 5' end labeling and clean-up of small RNA or DNA probes. For maximal convenience, the kit also contains reagents to prepare small radiolabeled RNA size markers (Decade™ Markers; 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, and 10 nt) and single-nucleotide RNA ladders.

One advantage of end labeling chemically synthesized oligonucleotides is that there is typically no need for gel purification of probes for use in the mirVana™ miRNA Detection Kit or RPA procedures, as is often required for probes prepared by in vitro transcription. However, there is usually a need for removal of unincorporated label. The purification columns included in the mirVana Probe & Marker Kit allow rapid and efficient clean up of 5' end labeled RNA probes.

Radiolabeled probes prepared with the mirVana Probe & Marker Kit have been successfully used for the detection of miRNA by Northern blot and by solution hybridization using the mirVana miRNA Detection Kit. DNA probes can also be efficiently labeled with the Probe & Marker Kit and used in many downstream applications.

How the Kit Works
Preparation of DNA and RNA probes or RNA markers starts with a phosphorylation reaction using T4 Polynucleotide Kinase and [gamma-³²P]ATP. If required, enzyme and unincorporated nucleotides can be subsequently removed by a rapid column purification procedure. After elution, the purified RNA is recovered in a concentrated aqueous solution.

To prepare the Decade Markers, first a phosphorylation reaction is performed with a 150 nt RNA transcript (included), then diluted into a cleavage reagent that generates the molecular weight marker set in a five minute room temperature reaction. For exact determination of RNA size, a single ribonucleotide ladder can also be generated using any purified 5' end labeled RNA probe in a ~10 min alkaline hydrolysis reaction following a protocol included in the Instruction Manual.

Note: The procedure was developed for desalted or more highly purified DNA and RNA oligonucleotides. However, any nucleic acid with a 5'-OH (e.g. DNA and RNA oligonucleotides, PCR products) can be labeled using this kit. Nucleic acids with a 5'-PO₄ (e.g. DNA cut by most restriction enzymes, in vitro transcribed RNAs, or small RNAs purified from biological samples) are not appropriate substrates unless the phosphate group is removed enzymatically.

Complete Kit for Probe and Marker Preparation
Each mirVana Probe & Marker Kit includes reagents for 30 end labeling reactions and 10 Decade Marker labeling reactions. In addition to T4 polynucleotide kinase and reaction buffer, the kit includes Purification Columns and associated wash and elution buffer, control RNA and DNA probes, and Gel Loading Buffer II. In addition, the kit includes Decade Marker RNA and Cleavage Reagent, as well as Alkaline Hydrolysis Buffer, for preparing RNA size markers and single-nucleotide RNA ladders, respectively.

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