• Sensitive — Detect miRNA or siRNA in as little as 50 ng total RNA
  
  
• Specific — Extremely low backgrounds
  
  
• Simple and fast — Single tube procedure eliminates the need to transfer to membrane for hybridization
  
  
• Multiple target detection — Detect multiple small RNAs and mRNAs in the same sample
  
  

The patented mirVana™ miRNA Detection Kit provides a fast and sensitive method for detecting small RNAs. The assay is 100–500 times more sensitive than Northern analysis and is able to detect as little as 10 attomoles (10^-17 mol) of target RNA. In addition, the kit can be used to simultaneously detect several small RNAs of the same size, or both small RNA and longer RNA species in the same sample. This means siRNA and its target mRNA can now be quantified in the same sample.

Rapid Procedure Based on Solution Hybridization
The mirVana miRNA Detection Kit is based on a simple solution hybridization principle (see top figure at right). The RNA sample containing the target RNA(s) of interest is simply mixed with one or more radiolabeled RNA probes and the included Hybridization Buffer. After heat denaturation, the mixture is incubated at 42°C to hybridize the probe to its complement. Unhybridized RNA and excess probe are then removed by a rapid ribonuclease digestion step. The hybridized, protected RNA fragments are recovered using Ambion’s patented single step technology for simultaneous ribonuclease inactivation and nucleic acid precipitation. RNA samples are then resuspended and analyzed on a denaturing polyacrylamide gel. The entire procedure has been specifically developed to provide optimal sensitivity and specificity with short antisense probes, and is ideal for detecting small RNA molecules such as siRNA or miRNA.

Detecting Multiple Small RNAs in the Same Sample
The mirVana miRNA Detection Kit can be used to detect multiple small RNAs, or one or more small RNAs and messenger RNAs in the same sample. To detect two small RNAs of the same size, one of the two probes is designed to have four A residues between the complementary region and the leader sequence. The leader sequence is designed to be cleaved by the RNases used in the assay and is present to distinguish protected from unprotected probe. The additional A residues adjacent to the leader sequences cannot be cleaved by the RNase mixture, and therefore increase the size of the protected fragment by four nucleotides. The ability of the mirVana miRNA Detection Kit to detect multiple similarly sized small RNA molecules gives it a distinct advantage over Northern analysis.

Simultaneous Detection of siRNA Expression and Target Gene Knockdown
Because multiple RNA species of the same or differing size can be detected in the same sample, the mirVana miRNA Detection Kit is ideal for correlating siRNA expression levels with target mRNA knockdown. An example of this application is shown in the bottom figure at right. This figure demonstrates that GAPDH mRNA levels were reduced in cells expressing a GAPDH siRNA, but not in cells expressing a control siRNA. The ability to correlate siRNA levels with targeted gene silencing is important for development of improved siRNA delivery systems and siRNA expression vectors.

Complete Kit with Included Controls
Each mirVana miRNA Detection Kit includes reagents for 100 reactions. The kit also contains a control transcription template and Probe Elution Buffer to prepare and gel purify a 32 nt RNA probe specific for miR-16 miRNA. When used with the provided mouse Kidney Total RNA, this probe generates a 22 nt protected fragment. The kit also comes with a detailed Instruction Manual that includes extensive information on experimental set up and probe design/preparation.

To prepare short radiolabeled RNA probes for use with this kit, please see the mirVana miRNA Probe Construction Kit (for probe synthesis and labeling by in vitro transcription) or the mirVana Probe & Marker Kit (for 5' end labeling of oligonucleotide probes and the included Decade Markers).

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