( A oligo (28 nt) was labeled and purifie...)Rapidly Prepare Probes and Markers for Small RNA Analysis

A oligo (28 nt) was labeled and purified with the mirVana Probe & Marker Kit, and 5 x 10⁴ cpm (~10 fmol) were used per solution hybridization assay with the mirVana miRNA Detection Kit. Samples were analyzed on a 15% denaturing polyacrylamide gel. Autoradiography was for 16 hours at 80C.

Easy Preparation of Probes and Small RNA Markers
Preparation of RNA markers and DNA or RNA probes starts with a phosphorylation reaction using T4 Polynucleotide Kinase and Kinase Buffer supplied with the kit, and [gamma-³²P]ATP. If required, Kinase Buffer, enzyme, and unincorporated nucleotides can be removed from the labeling reaction by a rapid column purification procedure. After elution, the purified probe is recovered in a concentrated form in aqueous solution. To prepare the Decade Markers, the phosphorylation reaction is performed with a 150 nt RNA transcript (provided), then diluted into a cleavage reagent that generates the molecular weight marker set in a 5 minute, room temperature reaction. For exact determination of RNA size, a single ribonucleotide ladder (Figure 1) can also be generated using any purified 5' end labeled RNA probe in a ~10 minute alkaline hydrolysis reaction.

Complete Kit for Small RNA Analysis
The mirVana Probe & Marker Kit includes enough reagents and Purification Cartridges for 30 labeling reactions and 30 probe purifications. Reagents for 10 preparative reac
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