( Radiolabeled RNA prob...)Rapidly Prepare Probes and Markers for Small RNA Analysis

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 g 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 1. Examples of Probes and Markers. Decade Markers, Control DNA (5S rRNA, 16 nt), and Control RNA (mir-16, 28 nt) probes were labeled with the mirVana Probe & Marker Kit as per the protocol. The RNA markers, mir-16 RNA probe, and a 32 nt long RNA probe synthesized by in vitro transcription (IVT) with the mirVana miRNA Probe Construction Kit, were then purified with the provided Purification Cartridges. The purified markers, unpurified DNA-probe (5S rRNA), purified mir-16 RNA probe, single-nucleotide RNA ladder prepared using the purified mir-16 probe, and purified IVT probe were resolved on a 15% denaturing polyacrylamide gel.

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 RN
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