Rapidly Prepare Probes and Markers for Small RNA Analysis
mirVana™ Probe & Marker Kit
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The new mirVana Probe & Marker Kit provides a complete solution to quickly prepare and purify high specific activity radiolabeled RNA probes and small RNA markers such as Decade Markers (Figure 1). These tools are ideal for investigating a variety of small RNA molecules such as transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), and micro RNA (miRNA).
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.
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 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 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.
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 reactions of Decade Markers are also supplied, providing RNA markers for 100-500 experiments. The kit also includes 2 functional oligonucleotide positive controls: a DNA probe specific for 5S rRNA and an RNA probe specific for miR-16 miRNA (Figure 1). Each control labeling reaction will produce enough probe for about 10 Northerns or hundreds of solution hybridization assays (RNA probe only). Gel Loading Buffer, RNase-free Water, Alkaline Hydrolysis Buffer and a manual with detailed protocol and additional procedures are also provided.
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