mirVana™ miRNA Probe Construction Kit

Prepare siRNA and miRNA Probes in Just 1 Hour

mirVana™ miRNA Probe Construction Kit

Prepare your probe in less than 1 hour
Ideal for siRNA/miRNA studies
Synthesize high specific activity RNA probes for RPA, Northern, Southern, or in situ hybridization
No cloning required

With the mirVana miRNA Probe Construction Kit, short (<100 nt) radiolabeled or nonisotopically labeled RNA probes can be generated in less than 1 hour. These probes are ideal for the investigation of a variety of small RNAs. Radiolabeled probes prepared with the mirVana miRNA Probe Construction Kit have been successfully used for the detection of small nuclear RNA (snRNA), small interfering RNA (siRNA), micro RNA (miRNA), and mRNA by RPAs, Northerns and in situ hybridization. Nonisotopically labeled probes generated with the kit have also been used to study the distribution of miRNA or mRNA in tissues by in situ hybridization (See Analysis of miRNA Distribution in Mouse Brain, right).

RNA Probe Synthesis Made Fast, Simple and Inexpensive

The mirVana miRNA Probe Construction Kit includes reagents for preparation of in vitro transcription templates as well as reagents for in vitro transcription. For template preparation, all you need to supply is a short, inexpensive DNA oligonucleotide specific for the target of interest that also includes an 8 base sequence complementary to the 3' end of the T7 promoter primer included in the kit. This oligonucleotide is annealed to the provided T7 promoter primer, and a 30 minute fill-in reaction with Klenow fragment generates the double-stranded transcription template. The resulting template is simply mixed with the provided T7 RNA Polymerase, rNTPs, and Transcription Buffer along with your choice of labeled rNTP. The reaction is then incubated for as little as 10 minutes at 37°C to generate labeled probe.

Complete Kit for Probe Preparation

The kit includes enough reagents to prepare 30 dsDNA templates and to perform 30 in vitro transcription reactions. Each template construction reaction supplies sufficient template for up to 20 transcription reactions. The kit also includes a functional positive control, DNase I to remove residual DNA template, and Probe Elution Buffer to gel purify RNA transcripts.

Figure 1. miR-16 and miR-22 Expression in Mouse Brain. In situ hybridization was performed on coronal mouse forebrain sections. In situ hybridized cells in the mouse brain cortex (A,B,D,E; 20X magnification) and the head of the caudate nucleus (C,F; 40X magnification) are indicated by arrows. Nonisotopically labeled probes were prepared with Ambion's MAXIscript™ Kit (1.5 kb probe) or with the mirVana miRNA Probe Construction Kit (32 nt long probes, 22 nt target specific sequence).

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Analysis of miRNA Distribution in Mouse Brain Although the tissue-specific expression pattern of miRNA is becoming well documented, little or no data is available about their distribution within cells or tissues. This lack of information mainly stems from the purported difficulties in using very small probes for in situ hybridization studies.

Ambion's scientists have optimized procedures for the sensitive and specific detection of mRNA and miRNA by in situ hybridization with small probes. As shown in Figure 1 (below left), the same cell-specific expression of VIP mRNA was observed in mouse cortex with a 1.5 kb probe as with a 22 nt long probe prepared with the mirVana miRNA Probe Construction Kit (Panels A and B). This result demonstrated that the small probes yielded specific signal.

Using additional small RNA probes, miR-16 and miR-22 miRNAs were found to be widely distributed in the cortex layers 2 and 3 (Panels B and E). Consistent with this result, high levels of miR-16 and miR-22 expression were also detected in mouse brain total RNA by hybridization in solution with the mirVana miRNA Detection Kit (data not shown). However, differences between the miR-16 and miR-22 expression patterns were noted within the brain. For example, only very few miR-16 positive cells were observed in the head of the caudate nucleus (Panels C and F). The staining pattern in this area was characteristic of cytoplasmic subcellular localization for both miRNA targets.

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