Rapidly Prepare Probes and Markers for Small RNA Analysis
mirVana™ Probe & Marker Kit
The purification system included in the kit allows fast and efficient clean up of 5' end labeled probes. The Purification Cartridges can also be used to clean up small probes prepared by in vitro transcription (e.g. with the mirVana™ miRNA Probe Construction Kit, see Figure 1). Unlike RNA probes prepared by in vitro transcription, 5' labeled probes generally do not require gel purification prior to use in a sensitive assay such as solution hybridization.
Radiolabeled RNA probes prepared with the mirVana Probe & Marker Kit have been successfully used for the detection of miRNA by Northern blot, or by solution hybridization with the mirVana™ miRNA Detection Kit (Figure 2). In this experiment miR-124 was detected in as little as 10 ng of total mouse brain RNA. In contrast, no expression of this brain-specific miRNA was observed in 1.5 µg total RNA from HeLa cells. DNA probes can also be efficiently labeled with the mirVana Probe & Marker Kit and used in various downstream applications, such as primer extension or Northern blot (data not shown).
Figure 2. Sensitive and Specific Detection of miR-124 miRNA. miR-124 (22 nt) was detected in the indicated amount of total RNA purified from two different mouse brains or two independent HeLa cell samples with the mirVana™ miRNA Isolation Kit. 2.5 pmol of HPLC-purified RNA 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 80°C.